Polynucleotides related to pancreatic disease

ABSTRACT

The present invention is based on the discovery of polynucleotides that represent novel genes that are differentially expressed in pancreatic disease, e.g., pancreatic cancer, dysplasia, pancreatitis, or diabetes. The invention features methods of identifying cells affected by such pancreatic diseases by detection of a gene product encoded by such differentially expressed genes, as well as methods of modulating expression of such gene products to effect therapy (e.g., to decrease growth and/or affect abnormal characteristics of cancerous or dysplastic pancreatic cells.)

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of prior U.S. ProvisionalApplication Serial No. 5 60/118,302, filed Feb. 2, 1999, whichapplication is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] The invention relates to genes differentially expressedpancreatic disease, in particular, pancreatic cancer, dysplasia, anddiabetes. More specifically, it relates to polynucleotides that aredifferentially regulated in pancreatic cancer and dysplasia.

BACKGROUND OF THE INVENTION

[0003] Cancer of the pancreas is the fifth leading cause of cancer deathin the United States. According to the American Cancer Society,approximately 28,000 people will die of pancreatic cancer in the UnitedStates in 1998. The pancreas is a tongue-shaped glandular organ composedof both endocrine and exocrine gland portions, as well as ducts thatconnect the pancreas to the bile duct and small intestine. The endocrineportion of the pancreas secretes hormones, such as insulin and glucagonwhich are involved in blood sugar regulation, into the bloodstream. Theexocrine portion of the pancreas produces pancreatic enzymes involved inthe digestion of fats and proteins; these enzymes are delivered to thebile duct and into the small intestine.

[0004] Little is known about the causes of pancreatic cancer, althoughit is apparent that a high risk of developing pancreatic cancer, withouta corresponding increase in the risk of developing other cancers, may bepassed along in some families. Cigarette smoking is the mostconsistently observed non-genetic risk factor for tumor development,with the disease being two to three times more common in heavy smokersthan in nonsmokers. However, it is uncertain whether this apparentassociation reflects a direct carcinogenic effect of metabolites ofcigarette smoke or whether an as yet undefined exposure is responsiblefor the observed enhanced risk. Both chronic pancreatitis andlong-standing diabetes mellitus have each been linked to an increasedrisk of pancreatic cancer. Mutations in K-ras genes have been found inmore than 85 percent of specimens of human pancreatic cancer. Thedevelopment of pancreatic cancer has also been associated with amutation of the p16.^(INK4) gene located on chromosome 9p21, a genewhich is also implicated in the pathogenesis of cutaneous malignantmelanoma.

[0005] Overall, pancreatic cancers occur twice as frequently in thepancreatic head (about 70% of cases) as in the body (about 20%) or tail(about 10%). Pancreatic adenocarcinomas usually begin in the ducts ofthe pancreas, but may sometimes develop from the acinar cells. Greaterthan 90% of pancreatic cancers are ductal adenocarcinomas, with theremaining 5 to 10% being islet cell tumors. Cancers of the exocrinecells of the pancreas are usually adenocarcinomas (about 95%). Lesscommon cancers of the exocrine pancreas include adenosquamouscarcinomas, squamous cell carcinomas, and giant cell carcinomas.

[0006] The initial symptoms of pancreatic cancer are usually nonspecific(e.g., abdominal pain and weight loss) and are frequently disregarded.The deep anatomic location of the pancreas makes detection of smalllocalized tumors unlikely during the routine abdominal examination. Evenin patients with confirmed pancreatic cancer, an abdominal mass ispalpable in only 15-25% of cases. Diagnosis of pancreatic cancer isfurther complicated by the occurrence of dysplastic cells, i.e.,abnormal cells that are not cancerous. Thus, even a biopsy can result inan erroneous diagnosis. Biopsy diagnoses may also be complicated byother underlying pancreatic disorders such as diabetes or pancreatitis.Unfortunately, because pancreatic cancer is generally very aggressive,some 80-90% of patients have regional and distant metastases by the timethey are diagnosed and only 3% of the 24,000 patients annually diagnosedwith pancreatic cancer live more than 5 years after diagnosis.

[0007] Although early and accurate diagnosis can thus be extremelyimportant in treatment success, there are presently no reliablescreening tests for detecting pancreatic cancer in asymptomatic persons.Imaging procedures such as magnetic resonance imaging and computedtomography are too costly to use as routine screening tests, while moreaccurate tests such as endoscopic retrograde cholangiopancreatography(ERCP) and endoscopic ultrasound are inappropriate for screeningasymptomatic patients due to their invasiveness. Abdominalultrasonography is a noninvasive screening test, but there is littleinformation on the efficacy of abdominal ultrasound as a screening testfor pancreatic cancer in asymptomatic persons. In symptomatic patientswith suspected disease it has a reported sensitivity of 40-98% and aspecificity as high as 90-94%. Conventional ultrasonography is limitedby visualization difficulties in the presence of bowel gas or obesityand by its range of resolution (2-3 cm). Even tumors less than 2 cm indiameter are frequently associated with metastatic disease, thuslimiting the ability of ultrasound to detect early disease.

[0008] Most persons with pancreatic malignancy have elevated levels ofcertain serologic markers such as CA19-9, peanut agglutinin, pancreaticoncofetal antigen, DU-PAN-2, carcinoembryonic antigen,alpha-fetoprotein, CA-50, SPan-1, and tissue polypeptide antigen (Rhodeset al. (1990) Bailleres Clin. Gastroenterol. 4:833; Steinberg (1990) Am.J. Gastroenterol. 85:350; Satake et al. (1990) Int. J. Pancreatol: 7:25;Satake (1991) Int. J. Pancreatol: 9:93). None of these markers is,however, tumor specific or organ specific (Satake (1991), supra).Elevations of various serologic markers also occur in significantproportions of persons with benign gastrointestinal diseases ormalignancies other than pancreatic cancer (Carter (1990) Gut 31:494;Rhodes et al. (1990), supra; Satake et al. (1990), supra; Satake (1991),supra). Most of these markers have been studied exclusively in high-riskpopulations, such as symptomatic patients with suspected pancreaticcancer. CA 19-9 has probably achieved the widest acceptance as aserodiagnostic test for pancreatic carcinoma in symptomatic patients,with an overall sensitivity of approximately 80% (68-93%) andspecificity of 90% (73-100%); sensitivity was highest in patients withmore advanced disease (Steinberg (1990), supra; Satake et al. (1990),supra). Among healthy subjects, CA19-9 has good specificity (94-99%)(DelVillano et al. (1983) Clin. Chem. 29:549; Ritts et al. (1984) Int.J. Cancer 33:339; Fabris et al. (1988) Am. J. Gastroentrol. 83:549) butnevertheless generates a large proportion of false-positive results dueto the very low prevalence of pancreatic cancer in the generalpopulation (Frebourg et al. (1988) Cancer 62:2287; Homma et al. (1991)Int. J. Pancreatol. 9:119). The predictive value of a positive testcould be improved if a population at substantially higher risk could beidentified. Diabetes mellitus in older adult patients might be useful asa marker for a population at high risk of having pancreatic cancer.Cohort studies have reported incidences of pancreatic cancer amongdiabetic patients ranging from 51 to 166/100,000 person-years (Everhartet al. (19950 JAMA 273:1605).

[0009] The inadequacies of conventional diagnostic methods forpancreatic cancer highlight the need for diagnostic and therapeuticmethods and compositions, as well as for a better understanding of thedisease to provide the basis for more rationale and more quicklyresponsive therapy. The fact that some patients suffer from combinationsof pancreatic cancer, dysplasia, and/or diabetes further complicatesdiagnosis and rationale therapy design. The present invention addressesthis need by providing nucleotide sequence that are differentiallyexpressed in these diseases.

[0010] Relevant Literature

[0011] A review of diagnostic methods available for pancreatic cancer isprovided in Bramhall (1998) Int. J. Pancreatol. 23:83; Friess et al.(1997) Digestion 58:557; and Lemoine (1997) Digestion 58:550; as well asat the National Cancer Institute web sitehttp://cancernet.nci.nih.gov/clinpdq/soa/Pancreatic_cancer_Physician.html.

[0012] Expression analysis using nucleic acid arrays is reviewed byRamsay (1998) Nat. Biotech. 16:40-44. Methods for creating microarraysof biological samples, such as arrays of DNA samples to be used in DNAhybridization assays, are described in PCT publication no. WO 95/35505,published Dec. 28, 1995; U.S. Pat. No. 5,445,934; Drmanac et al.,Science 260:1649; and Yershov et al. (1996) Genetics 93:4913.

[0013] Quantitative monitoring of gene expression patterns with acomplementary DNA microarray is described in Schena et al. (1995)Science 270:467. DeRisi et al. (1997) Science 270:680-686 explore geneexpression on a genomic scale. Analysis of gene expression patterns inhuman cancer using a cDNA microarray is described in DeRisi et al.(1996) Nat. Genet. 14:457.

[0014] Use of differential display to identify differential geneexpression is described in, for example, U.S. Pat. No. 5,776,683; andU.S. Pat. No. 5,807,680.

[0015] Methods for preparation of substrate matrices (e.g., arrays),design of oligonucleotides for use with such matrices, labeling ofprobes, hybridization conditions, scanning of hybridized matrices, andanalysis of patterns generated, including comparison analysis, aredescribed in, for example, U.S. Pat. No. 5,800,992.

SUMMARY OF THE INVENTION

[0016] The present invention is based on the discovery ofpolynucleotides that represent novel genes that are differentiallyexpressed in pancreatic disease, e.g., pancreatic cancer, dysplasia,pancreatitis, or diabetes. The invention features methods of identifyingcells affected by such pancreatic diseases by detection of a geneproduct encoded by such differentially expressed genes, as well asmethods of modulating expression of such gene products to effect therapy(e.g., to decrease growth and/or affect abnormal characteristics ofcancerous or dysplastic pancreatic cells.)

[0017] Accordingly, in one aspect the invention features a library ofdifferentially expressed genes, where the library includes the sequenceinformation of at least one of the polynucleotides of SEQ ID NOS: 1-6.The library may be provided as a nucleic acid array or acomputer-readable format, and may include relative amounts of thepolynucleotides of SEQ ID NOS: 1-6, where the relative amounts arerepresentative of relative amounts of the polynucleotides found in adiseased pancreatic cell.

[0018] The invention also features an isolated polynucleotide having asequence of at least 90% sequence identity to an identifying sequence ofSEQ ID NOS: 1-6 or degenerate variants thereof. In related aspects, theinvention features arrays and recombinant host cells having apolynucleotide of the invention. In one embodiment the polynucleotideincludes the nucleotide sequence of an insert contained in one of theclones HX2134-4, HX2144-1, HX2145-3, HX2162-3, HX2166-6, or HX2192-1,which have been deposited as ATCC accession number 98896.

[0019] In another aspect the invention features an isolated polypeptideencoded by a differentially express gene of the invention, as well asantibodies that specifically bind such polypeptides.

[0020] The invention also features a method of identifying a cancerouspancreatic cell, where the method involves detecting at least onedifferentially expressed gene product, where the gene product is encodedby a gene having a sequence of SEQ ID NOS: 1-6 in a test sample, wherethe test sample is derived from a test cell suspected of being acancerous pancreatic cell, and comparing an amount of the detecteddifferentially expressed gene product with an amount of thedifferentially expressed gene product in a control sample, where thecontrol sample is derived from a cancerous pancreatic cell. Detection ofan amount of the differentially expressed gene product in the testsample that is similar to an amount of the gene product in the controlsample indicates that the test cells is a cancerous pancreatic cell. Inone embodiment, detection is accomplished by hybridization of the testsample to a reference array, wherein the reference array comprises anidentifying sequence of at least one of SEQ ID NOS: 1-6.

[0021] In another aspect, the invention features therapeuticcompositions having an active agent for modulation of expression of agene differentially expressed in cancerous or dysplastic pancreaticcells. In specific embodiments, the active agent of the therapeuticcomposition effects a decrease in biological activity of a gene productencoded by a gene having a sequence of SEQ ID NO: 2, effects an increasein biological activity of a gene product encoded by a gene having asequence of SEQ ID NO: 6, effects an increase in biological activity ofa gene product encoded by a gene having a sequence of SEQ ID NOS: 1 or3, or effects a decrease in biological activity of a gene productencoded by a gene having a sequence of SEQ ID NOS: 4 or 5.

[0022] A primary object of the invention is to provide differentiallyexpressed polynucleotides and fragments thereof that are useful indiagnosis of pancreatic disease, as well as in rational drug and therapydesign.

[0023] These and other objects of the invention are provided by one ormore of the embodiments described below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0025] In this specification and the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

[0026] Definitions

[0027] The term “differentially expressed gene” is intended to encompassa polynucleotide that may include an open reading frame encoding a geneproduct (e.g., a polypeptide), as well as introns of such genes andadjacent 5′ and 3′ non-coding nucleotide sequences involved in theregulation of expression, up to about 20 kb beyond the coding region,but possibly further in either direction. The gene may be introducedinto an appropriate vector for extrachromosomal maintenance or forintegration into a host genome. In general, a difference in expressionlevel associated with a decrease in expression level of at least about25%, usually at least about 50% to 75%, more usually at least about 90%or more is indicative of a differentially expressed gene of interest,i.e., a gene that is underexpressed or down-regulated in the test samplerelative to a control sample. Furthermore, a difference in expressionlevel associated with an increase in expression of at least about 25%,usually at least about 50% to 75%, more usually at least about 90% andmay be at least about 1½-fold, usually at least about 2-fold to about10-fold, and may be about 100-fold to about 1,000-fold increase relativeto a control sample is indicative of a differentially expressed gene ofinterest, i.e., an overexpressed or up-regulated gene.

[0028] “Differentially expressed polynucleotide” as used herein means anucleic acid molecule (RNA or DNA) having a sequence that represents adifferentially expressed gene, e.g., the differentially expressedpolynucleotide comprises a sequence (e.g., an open reading frameencoding a gene product) that uniquely identifies a differentiallyexpressed gene so that detection of the differentially expressedpolynucleotide in a sample is correlated with the presence of adifferentially expressed gene in a sample. “Differentially expressedpolynucleotides” is also meant to encompass fragments of the disclosedpolynucleotides, e.g., fragments retaining biological activity, as wellas nucleic acids that are homologous, substantially similar, orsubstantially identical (e.g., having about 90% sequence identity) tothe disclosed polynucleotides.

[0029] “Reference sequences” or “reference polynucleotides” as usedherein in the context of differential gene expressive analysis anddiagnosis refers to a selected set of polynucleotides, which selectedset includes at least one or more of the differentially expressedpolynucleotides described herein. A plurality of reference sequences,preferably comprising positive and negative control sequences, may beincluded as reference sequences. Additional reference sequences that maybe used as reference sequences are found in Genbank, Unigene, and othernucleotide sequence databases (including, e.g., expressed sequence tag(EST), partial, and full-length sequences).

[0030] “Reference array” means an array having reference sequences foruse in hybridization with a sample, where the reference sequencesinclude all, at least one of, or any subset of the differentiallyexpressed polynucleotides listed in Table 1. Usually such an array willinclude at least 3 different reference sequences, and may include anyone or all of the provided differentially expressed sequences. Arrays ofinterest may further comprise sequences, including polymorphisms, ofother genetic sequences, particularly other sequences of interest forscreening for a pancreatic disorder (e.g., pancreatic cancer, pancreaticdysplasia, pancreatitis, diabetes syndromes, and the like). Theoligonucleotide sequence on the array will usually be at least about 12nt in length, and may be of about the length of the sequences providedin Table 1, or may extend into the flanking regions to generatefragments of 100 nt to 200 nt in length or more.

[0031] A “reference expression pattern” or “REP” as used herein refersto the relative levels of expression of a selected set of genes,particularly of differentially expressed genes, that is associated witha selected cell type, e.g., a normal cell (e.g., normal pancreaticcell), a cancerous cell, a cell exposed to an environmental stimulus,and the like.

[0032] A “test expression pattern” or “TEP” refers to relative levels ofexpression of a selected set of genes, particularly of differentiallyexpressed genes, in a cell of a test sample (e.g., a cell of unknown orsuspected disease state, from which mRNA is isolated).

[0033] “Diagnosis” as used herein generally includes determination of asubject's susceptibility to a disease or disorder, determination as towhether a subject is presently affected by a disease or disorder, aswell as to the prognosis of a subject affected by a disease or disorder.The present invention encompasses diagnosis of subjects in the contextof pancreatic cancer (e.g., ductal adenocarcinoma or other pancreaticcancer, as well as any stage of such cancers (e.g., stages I to IV inseverity), pancreatic dysplasia, pancreatitis, and diabetes (e.g., TypeI or Type II diabetes).

[0034] “Pancreatic cancer” is meant to encompass benign or malignantforms of pancreatic cancer, as well as any particular type of cancerarising from cells of the pancreas (e.g., duct cell carcinoma, acinarcell carcinoma, papillary carcinoma, adenosquamous carcinoma,undifferentiated carcinoma, mucinous carcinoma, giant cell carcinoma,mixed type pancreatic cancer, small cell carcinoma, cystadenocarcinoma,unclassified pancreatic cancers, pancreatoblastoma, and papillary-cysticneoplasm, and the like.

[0035] “Pancreatitis” as used herein is meant to encompass chronicpancreatitis, acute pancreatitis, and pancreatic abscesses associatedwith pancreatic inflammation.

[0036] “Subjects” or “patients” as used herein is meant to encompass anysubject or patient amenable to application of the diagnostic and/ortherapeutic methods of the invention. Mammalian subjects and patients,particularly human subjects or patients are of particular interest.

[0037] “Sample” or “biological sample” are generally used to refer tosamples of biological fluids or tissues, particularly samples obtainedfrom pancreatic tissues, especially from pancreatic cells of the typeassociated with the disease for which the diagnostic application isdesigned (e.g., ductal adenocarcinoma), and the like. “Samples” is alsomeant to encompass derivatives and fractions of such samples (e.g., celllysates). Where the sample is solid tissue, the cells of the tissue maybe dissociated or tissue sections may be analyzed.

[0038] Overview of the Invention

[0039] In general, the invention is based on the discovery ofpolynucleotides that represent genes that are differentially expressedin pancreatic cells associated with pancreatic disease, particularlypancreatic cancer, dysplasia, pancreatitis, and/or diabetes.Differential expression of genes in pancreatic cells affected withcancer is determined by, for example, detecting genes expressed in acancerous pancreatic cell, and comparing the level of gene expression toexpression of those same genes in a normal pancreatic cell (i.e., apancreatic cell that is not affected by a pancreatic cancer) and/or adysplastic pancreatic cell.

[0040] The differentially expressed polynucleotides described hereinwere identified using differential displays of samples from normalpancreatic cells, dysplastic pancreatic cells, cancerous pancreaticcells, pancreatic cells from a subject affected by pancreatitis, andpancreatic cells from a subject affected by diabetes (Type I or TypeII). The sequence of specific polynucleotides that representdifferentially expressed genes of the present invention are shown in SEQID NOS: 1-6. Differential expression of the genes represented by thesepolynucleotides was observed to be as follows:

[0041] 1. SEQ ID NOS: 1 and 3 are expressed at a relatively lower level(i.e., down-regulated) in a dysplastic pancreatic cell;

[0042] 2. SEQ ID NO: 2 is expressed at a relatively higher level (i.e.,up-regulated) in a cancerous pancreatic cell;

[0043] 3. SEQ ID NOS: 4 and 5 are expressed at a relatively higher levelin a dysplastic pancreatic cell; and

[0044] 4. SEQ ID NO: 6 is expressed at a relatively lower level in bothpancreatic cells affected by dysplasia and cancerous pancreatic cells.

[0045] The invention will now be described in further detail.

[0046] Nucleic Acid Compositions

[0047] The invention features polynucleotides that are differentiallyexpressed in pancreatic disease (e.g., cancer, dysplasia, diabetes, orpancreatitis). Novel nucleic acid compositions of the invention ofparticular interest comprise a sequence set forth in any one of SEQ IDNOS: 1-6 or an identifying sequence thereof. An “identifying sequence”is a contiguous sequence of residues at least about 10 nt to about 20 ntin length, usually at least about 50 nt to about 100 nt in length, thatuniquely identifies a polynucleotide sequence, e.g., exhibits less than90%, usually less than about 80% to about 85% sequence identity to anycontiguous nucleotide sequence of more than about 20 nt. Thus, thesubject novel nucleic acid compositions include full length cDNAs ormRNAs that include an identifying sequence of contiguous nucleotidesfrom any one of SEQ ID NOS: 1-6, as described above.

[0048] The polynucleotides of the invention also include naturallyoccurring variants of the nucleotide sequences (e.g., degeneratevariants, allelic variants, etc.). Allelic variants of thepolynucleotides of the invention are identified by hybridization ofputative allelic variants with nucleotide sequences disclosed hereinunder stringent conditions. Nucleic acids having sequence similarity aredetected by hybridization under low stringency conditions, for example,at 50° C. and 10×SSC (0.9 M saline/0.09 M sodium citrate) and remainbound when subjected to washing at 55° C. in 1×SSC. Sequence identitycan be determined by hybridization under stringent conditions, forexample, at 50° C. or higher and 0.1×SSC (9 mM saline/0.9 mM sodiumcitrate). Hybridization methods and conditions are well known in theart, see, e.g., U.S. Pat. No. 5,707,829. Nucleic acids that aresubstantially identical to the provided polynucleotide sequences, e.g.allelic variants, genetically altered versions of the gene, etc.,generally bind to the provided polynucleotide sequences (SEQ ID NOS:1-6) under stringent hybridization conditions. In general, allelicvariants contain 15-25% base pair mismatches, and may contain as littleas even 5-15%, or 2-5%, or 1-2% base pair mismatches, as well as asingle base-pair mismatch.

[0049] The invention also encompasses homologs corresponding to thepolynucleotides of SEQ ID NOS: 1-6, where the source of homologous genesmay be any mammalian species, e.g., primate species, particularly human;rodents, such as rats, canines, felines, bovines, ovines, equines,yeast, nematodes, etc. Between mammalian species, e.g., human and mouse,homologs have substantial sequence similarity, e.g. at least 75%sequence identity, usually at least 90%, more usually at least 95%between nucleotide sequences. Sequence similarity is calculated based ona reference sequence, which may be a subset of a larger sequence, suchas a conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 contiguous nt long, moreusually at least about 30 nt long, and may extend to the completesequence that is being compared. Algorithms for sequence analysis areknown in the art, such as BLAST, described in Altschul et al. (1990), J.Mol. Biol. 215:403-10. In general, variants of the invention have asequence identity greater than at least about 65%, preferably at leastabout 75%, more preferably at least about 85%, and may be greater thanat least about 90% or more as determined by the Smith-Waterman homologysearch algorithm as implemented in MPSRCH program (Oxford Molecular)using an affine gap search with the following search parameters: gapopen penalty: 12; and gap extension penalty: 1. The sequences providedherein are essential for recognizing related and homologouspolynucleotides in database searches.

[0050] The subject nucleic acids may be cDNAs or genomic DNAs, as wellas fragments thereof, particularly fragments that encode a biologicallyactive gene product and/or are useful in the methods disclosed herein(e.g., in diagnosis, as a unique identifier of a differentiallyexpressed gene of interest, etc.). The term “cDNA” as used herein isintended to include all nucleic acids that share the arrangement ofsequence elements found in native mature mRNA species, where sequenceelements are exons and 3′ and 5′ non-coding regions. Normally mRNAspecies have contiguous exons, with the intervening introns, whenpresent, being removed by nuclear RNA splicing, to create a continuousopen reading frame encoding a polypeptide of the invention.

[0051] A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include the 3′ and 5′untranslated regions found in the mature mRNA. It may further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ and 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ and 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue, stage-specific, or disease-state specificexpression.

[0052] The nucleic acid compositions of the subject invention may encodeall or a part of the subject differentially expressed polypeptides.Double or single stranded fragments may be obtained from the DNAsequence by chemically synthesizing oligonucleotides in accordance withconventional methods, by restriction enzyme digestion, by PCRamplification, etc. Isolated polynucleotides and polynucleotidefragments of the invention comprise at least 10, 11, 12, 15, 18, 20, 25,30, 35, 40, 45, 50, 60, 70, 74, 80, 90, 100, 125, 150, 154, 175, 200,250, 300, or 350 contiguous nucleotides selected from the polynucleotidesequences as shown in SEQ ID NOS: 1-6. For the most part, fragments willbe of at least 15 nt, usually at least 18 nt or 25 nt, and may be atleast about 50 contiguous nt in length. In a preferred embodiment, thepolynucleotide molecules comprise a contiguous sequence of at leasttwelve nucleotides selected from the group consisting of thepolynucleotides shown in SEQ ID NOS: 1-6.

[0053] Probes specific to the polynucleotides of the invention may begenerated using the polynucleotide sequences disclosed in SEQ ID NOS:1-6. The probes are preferably at least a 12, 14, 16, 18, 20, 22, 24, or25 nucleotide fragment of a corresponding contiguous sequence of SEQ IDNOS: 1-6, and can be less than 2, 1, 0.5, 0.1, or 0.05 kb in length. Theprobes can be synthesized chemically or can be generated from longerpolynucleotides using restriction enzymes. The probes can be labeled,for example, with a radioactive, biotinylated, or fluorescent tag.

[0054] The differentially expressed polynucleotides of the subjectinvention are isolated and obtained in substantial purity, generally asother than an intact chromosome. Usually, the DNA will be obtainedsubstantially free of other naturally-occurring nucleic acid sequences,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant”, e.g., flanked by one or more nucleotideswith which it is not normally associated on a naturally occurringchromosome.

[0055] The polynucleotides of the invention can be provided as linear orwithin a circular molecules. They can be on autonomously replicatingmolecules (vectors) or on molecules without replication sequences. Theycan be regulated by their own or by other regulatory sequences, as isknown in the art. The polynucleotides of the invention can be introducedinto suitable host cells using a variety of techniques which areavailable in the art, such as transferrin polycation-mediated DNAtransfer, transfection with naked or encapsulated nucleic acids,liposome-mediated DNA transfer, intracellular transportation ofDNA-coated latex beads, protoplast fusion, viral infection,electroporation, gene gun, calcium phosphate-mediated transfection, andthe like.

[0056] The subject nucleic acid compositions can be used to, forexample, produce polypeptides, as probes for the detection of mRNA ofthe invention in samples or extracts of human cells, to generateadditional copies of the polynucleotides, to generate ribozymes orantisense oligonucleotides, and as single stranded DNA probes or astriple-strand forming oligonucleotides. The probes described herein canbe used to, for example, determine the presence or absence of thepolynucleotide sequences as shown in SEQ ID NOS: 1-6 or variants thereofin a sample.

[0057] Polypeptide Compositions

[0058] The subject invention also provides polypeptides encoded by adifferentially expressed polynucleotide of the invention, e.g, apolypeptide encoded by a polynucleotide having a sequence of any of SEQID NOS: 1-6. The term “polypeptide” composition as used herein refers toboth the full length polypeptide encoded by the recited polynucleotide,the polypeptide encoded by the gene represented by the recitedpolynucleotide, as well as portions or fragments thereof. “Polypeptides”also includes variants of the naturally occurring proteins, where suchvariants are homologous or substantially similar to the naturallyoccurring protein, and can be of an origin of the same or differentspecies as the naturally occurring protein (e.g., human, murine, or someother species that naturally expresses the recited polypeptide, usuallya mammalian species). In general, variant polypeptides have a sequencethat has at least about 80%, usually at least about 90%, and moreusually at least about 98% sequence identity with a differentiallyexpressed polypeptide of the invention, as measured by BLAST using theparameters described above. The variant polypeptides may be naturally ornon-naturally glycosylated, i.e., the polypeptide has a glycosylationpattern that differs from the glycosylation pattern found in thecorresponding naturally occurring protein.

[0059] The invention also encompasses homologs of the disclosedpolypeptides (or fragments thereof) where the homologs are isolated fromother species, i.e. other animal or plant species, where such homologs,usually mammalian species, e.g. rodents, such as mice, rats; domesticanimals, e.g., horse, cow, dog, cat; and humans. By homolog is meant apolypeptide having at least about 35%, usually at least about 40% andmore usually at least about 60% amino acid sequence identity aparticular differentially expressed protein as identified above, wheresequence identity is determined using the BLAST algorithm, with theparameters described supra.

[0060] In general, the polypeptides of the subject invention areprovided in a non-naturally occurring environment, e.g. are separatedfrom their naturally occurring environment. In certain embodiments, thesubject protein is present in a composition that is enriched for theprotein as compared to a control. As such, purified polypeptide isprovided, where by purified is meant that the protein is present in acomposition that is substantially free of non-differentially expressedpolypeptides, where by substantially free is meant that less than 90%,usually less than 60% and more usually less than 50% of the compositionis made up of non-differentially expressed polypeptides.

[0061] In certain embodiments of interest, the subject protein ispresent in a composition that is substantially free of the constituentsthat are present in its naturally occurring environment. For example, acomposition comprising a protein according to the subject invention inthis embodiment will be substantially, if not completely, free of thoseother biological constituents, such as proteins, carbohydrates, lipids,etc., with which it is present in its natural environment. As such,protein compositions of these embodiments will necessarily differ fromthose that are prepared by purifying the protein from a naturallyoccurring source, where at least trace amounts of the protein's naturalenvironment constituents will still be present in the compositionprepared from the naturally occurring source.

[0062] The proteins of the subject invention may also be present as anisolate, by which is meant that the protein is substantially free ofboth non-differentially expressed polypeptides and other naturallyoccurring biologic molecules, such as oligosaccharides, polynucleotidesand fragments thereof, and the like, where substantially free in thisinstance means that less than 70%, usually less than 60% and moreusually less than 50% of the composition containing the isolatedpolypeptide is a non-differentially expressed, naturally occurringbiological molecule. In certain embodiments, the protein is present insubstantially pure form, where by substantially pure form is meant atleast 95%, usually at least 97% and more usually at least 99% pure.

[0063] In addition to the naturally occurring proteins, polypeptidesthat vary from the naturally occurring differentially expressedpolypeptides are also provided. By differentially expressed polypeptidesis meant polypeptides having an amino acid sequence encoded by an openreading frame (ORF) of a differentially expressed gene, especially adifferentially expressed polynucleotide of the invention,polynucleotide, including the full length polypeptide and fragmentsthereof, particularly biologically active fragments and/or fragmentscorresponding to functional domains; and including fusions of thesubject polypeptides to other proteins or parts thereof. Fragments ofinterest will typically be at least about 10 aa to at least about 15 aain length, usually at least about 50 aa in length, and may be as long as300 aa in length or longer, but will usually not exceed about 1000 aa inlength, where the fragment will have a stretch of amino acids that isidentical to a differentially expressed polypeptide encoded by adifferentially expressed gene having a sequence of any of SEQ ID NOS:1-6, or a homolog thereof.

[0064] Fusion polypeptides encompassed by the present invention arecomposed of at least two protein segments. The first protein segmentconsists of at least six, eight, ten, twelve, fifteen, twenty or thirtycontiguous amino acids of a polypeptide sequence expressed from apolynucleotide sequence as shown in SEQ ID NOS: 1-6. The first proteinsegment is fused to a second protein segment by means of a peptide bond.The second protein segment can be a full-length protein or a fragment ofa protein of the same, similar, or different origin. The second proteinor protein fragment can be labeled with a detectable marker, such as aradioactive or fluorescent tag, or an enzyme that can generate adetectable product upon contact with a substrate (e.g.,P-galactosidase). A fusion protein can be used, for example, tofacilitate delivery to a particular location in a cell or tissue, invarious biochemical or immunological assays, such as the yeasttwo-hybrid technique, or as an immunogen. Techniques for making fusionproteins, either recombinantly or by covalently linking two proteinsegments, are well know in the art.

[0065] Preparation of Differentially Expressed Polypeptides

[0066] The subject polypeptides may be obtained from naturally occurringsources, but are preferably synthetically produced. Where obtained fromnaturally occurring sources, the source chosen will generally be apancreatic cell. The subject polypeptide compositions may besynthetically derived by expressing a recombinant gene encoding thepolypeptide of interest in a suitable host. In general, an expressioncassette in an expression vector is used for recombinant expression. Theexpression vector provides transcriptional and translational initiationregions, for inducible or constitutive expression of an operably linkedcoding region, and transcriptional and translational terminationregions. These control regions may be native to a selecteddifferentially expressed gene, or may be derived from exogenous sources.

[0067] Expression vectors generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding heterologous proteins. A selectablemarker operative in the expression host may be present. Expressionvectors may be used for the production of fusion proteins, where theexogenous fusion peptide provides additional functionality, i.e.increased protein synthesis, stability, reactivity with definedantisera, an enzyme marker, e.g. β-galactosidase, etc.

[0068] Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

[0069] The polypeptides may be expressed in prokaryotes or eukaryotes inaccordance with conventional ways, depending upon the purpose forexpression. For large scale production of the encoded protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, particularly mammals, e.g., COS 7cells, may be used as the expression host cells. In some situations, itis desirable to express the differentially expressed polynucleotide ineukaryotic cells, where the encoded polypeptide will benefit from nativefolding and post-translational modifications. Small peptides can also besynthesized in the laboratory. Polypeptides that are encoded by subsetsof the complete differentially expressed sequence may be used toidentify and investigate parts of the polypeptide important forfunction.

[0070] Where it is desirable to produce a polypeptide of the inventionin a yeast host cell, suitable expression systems in yeast may beselected from those described in Hinnen et al., Proc. Natl. Acad. Sci.USA (1978) 75: 1929; Ito et al., J. Bacteriol. (1983) 153:163; Kurtz etal., Mol. Cell Biol. (1986) 6: 142; Kunze et al., J. Basic Microbiol.(1985) 25:14 1; Gleeson et al., J. Gen. Microbiol. (1986) 132: 3459;Roggenkamp, et al., Mol. Gen. Genet. (1986) 202:302; Das et al., J.Bacteriol. (1984) 158: 1165; De Louvencourt et al., J. Bacteriol. (1983)154: 737; Van den Berg et al., BioTechnology (1990) 8: 135; Kunze etal., J. Basic Microbiol. (1985) 25:141; Cregg et al., Mol. Cell. Biol.(1985) 5:3376; U.S. Pat. No. 4,837,148; U.S. Pat. No. 4,929,555; Beachand Nurse, Nature (1981) 300:706; Davidow et al., Curr. Genet. (1985)10: 3 80; Gaillardin et al., Curr. Genet. (1985) 10: 49; Ballance etal., Biochem. Biophys. Res. Commun. (1983) 112: 284-289; Tilbum et al.,Gene (1983) 26:205-22 1; Yelton et al., Proc. Natl. Acad. Sci. USA(1984) 81:1470-1474; Kelly and Hynes, EMBO J. (1985) 4:475479; EP244,234; and WO 91/00357.

[0071] Expression of the polynucleotides of the invention in insects canbe accomplished as described in U.S. Pat. No. 4,745,051, Friesen et al.(1986) “The Regulation of Baculovirus Gene Expression” in: The MolecularBiology of Baculovirus (W. Doerfler, ed.); EP 127,839; EP 155,476; Vlaket al., J. Gen. Virol. (1988) 69: 765-776; Miller et al., Ann. Rev.Microbiol. (1988) 42: 177; Carbonell et al., Gene (1988) 73: 409; Maedaet al., Nature (1985) 315:592-594; Lebacq-Verheyden et al., Mol. Cell.Biol. 8: 3129; Smith et al., Proc. Natl. Acad. Sci. USA (1985) 82:8404;Miyajima et al., Gene (1987) 58: 273; and Martin et al., DNA (1988)7:99. Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts are described in Luckow et al.,BioTechnol. (1988) 6: 47-55; Miller et al., in Genetic Engineering(Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp.277-279; and Maeda et al., Nature, (1985) 315:592-594.

[0072] Mammalian expression of the polynucleotides of the invention canbe accomplished as described in Dijkema et al., EMBO 1 (1985) 4:76;Gorman et al., Proc. Natl. Acad. Sci. USA (1982) 79:6777; Boshart etal., Cell (1985) 41:521; and U.S. Pat. No. 4,399,216. Other features ofmammalian expression can be facilitated as described in Ham and Wallace,Meth. Enzymol. (1979) 58: 44; Barnes and Sato, Anal. Biochem. (1980)102:255; U.S. Pat. No. 4,767,704; U.S. Pat. No. 4,657,866; U.S. Pat. No.4,927,762; U.S. Pat. No. 4,560,655; WO 90/103430; WO 87/00195; and U.S.RE30,985.

[0073] Once the source of the polypeptide is identified and/or prepared,e.g. a transfected host expressing the protein is prepared, thepolypeptide is then purified to produce the desired composition. Anyconvenient protein purification procedures may be employed, wheresuitable protein purification methodologies are described in Guide toProtein Purification, (Deuthser ed.) (Academic Press, 1990). Forexample, a lysate may be prepared from the original source, e.g.naturally occurring cells or tissues that express the protein or theexpression host expressing the protein, and purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,and the like.

[0074] Antibodies

[0075] The present invention also provides antibodies that specificallybind a polypeptide encoded by a differentially expressed polynucleotideor gene of the invention. Suitable antibodies are obtained by immunizinga host animal with a differentially expressed polypeptide. Suitablenon-human host animals include mice, rats, sheep, goats, hamsters,rabbits, etc. The origin of the protein immunogen may be mouse, human,rat, monkey etc. The host animal will generally be a different speciesthan from which the immunogen was derived, e.g. human protein used toimmunize mice, etc.

[0076] The immunogen may comprise the complete protein, or immunogenicfragments and derivatives thereof. Immunogens may include nativepost-translation modifications, such as glycosylation. Immunogens areproduced in a variety of ways known in the art, e.g. expression ofcloned genes using conventional recombinant methods, isolation from HEC,etc.

[0077] Polyclonal antibodies can be prepared by first immunizing thehost animal with a polypeptide, where the polypeptide will preferably bein substantially pure form, comprising less than about 1% contaminant.The protein may be combined with an adjuvant, where suitable adjuvantsinclude alum, dextran, sulfate, large polymeric anions, oil & wateremulsions, e.g., Freund's adjuvant, Freund's complete adjuvant, and thelike. The protein may also be conjugated to synthetic carrier proteinsor synthetic antigens. A variety of hosts may be immunized to producethe polyclonal antibodies (e.g., rabbits, guinea pigs, rodents, e.g.mice, rats, sheep, goats, and the like). The protein is administered tothe host, usually intradermally, with an initial dosage followed by oneor more, usually at least two, additional boosters. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

[0078] Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. The antibody may be purified from the hybridoma cellsupernatants or ascites fluid by conventional techniques, e.g. affinitychromatography using, protein A sepharose, etc. Anti-differentiallyexpressed polypeptide antibody may also be produced as a single chain,instead of the normal multimeric structure. Single chain antibodies aredescribed in Jost et al. (1994) J. Biol. Chem. 269:26267-73, and others.

[0079] For in vivo use, particularly for injection into humans, it isdesirable to decrease the antigenicity of the antibody. An immuneresponse of a recipient against the blocking agent will potentiallydecrease the period of time that the therapy is effective. Methods ofhumanizing antibodies are known in the art. For example, the humanizedantibody may be the product of an animal having transgenic humanimmunoglobulin constant region genes (see for example WO 90/10077 and WO90/04036). Alternatively, the antibody of interest may be engineered byrecombinant DNA techniques to substitute the CH₁, CH₂, CH₃, hingedomains, and/or the framework domain with the corresponding humansequence (see WO 92/02190).

[0080] The use of Ig cDNA for construction of chimeric immunoglobulingenes is known in the art (Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439 and (1987) J. Immunol. 139:3521). mRNA is isolated from ahybridoma or other cell producing the antibody and used to produce cDNA.The cDNA of interest may be amplified by the polymerase chain reactionusing specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al. (1991) Sequences ofProteins of Immunological Interest, N.I.H. publication no. 91-3242.Human constant (C) region genes are readily available from known clones.The choice of isotype will be guided by the desired effector functions,such as complement fixation, or activity in antibody-dependent cellularcytotoxicity. Preferred isotypes are IgG₁, IgG₃ and IgG₄. Either of thehuman light chain constant regions, kappa or lambda, may be used. Thechimeric, humanized antibody is then expressed by conventional methods.Chimeric antibodies may be produced by use of an expression vector(e.g., plasmid, retroviral vector, YAC, EBV-derived episome, etc.), andwhich may use any promoter, particularly a strong promoter, includingretroviral LTRs, e.g. SV-40 early promoter, (Okayama et al. (1983) Mol.Cell. Bio. 3:280), Rous sarcoma virus LTR (Gorman et al. (1982) P.N.A.S.79:6777), and moloney murine leukemia virus LTR (Grosschedl et al.(1985) Cell 41:885); native Ig promoters, etc.

[0081] Antibody fragments, such as Fv, F(ab′)₂ and Fab may be preparedby cleavage of an intact Ig protein, e.g. by protease or chemicalcleavage. Alternatively, a truncated gene is designed. For example, achimeric gene encoding a portion of the F(ab′)₂ fragment would includeDNA sequences encoding the CH₁ domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

[0082] Uses of the Subject Polypeptide and Nucleic Acid Compositions

[0083] The compositions of the invention find use in a variety ofapplications, such as: (a) the identification of differentiallyexpressed gene homologs; (b) as a source of novel promoter elements; (c)the identification of naturally-occurring factors that regulateexpression; (d) as probes and primers in hybridization applications,e.g. PCR; (e) the identification of expression patterns in biologicalspecimens; (f) the preparation of cell or animal models fordifferentially expressed protein function; (g) the preparation of invitro models for function of differentially expressed polypeptides; etc.Exemplary applications are described below.

[0084] Identification of Homologs of Differentially ExpressedPolynucleotides.

[0085] Homologs of the differentially expressed polynucleotide of theinvention can be identified by any of a number of methods. For example,a fragment of the provided cDNA may be used as a hybridization probeagainst a cDNA library from the target organism of interest, where lowstringency conditions are used. The probe may be a large fragment, orone or more short degenerate probes. Nucleic acids having a region ofsubstantial identity to the provided differentially expressed nucleicacid sequences, e.g. allelic variants, genetically altered versions ofthe gene, etc., bind to the sequences of the invention under stringenthybridization conditions. By using probes, particularly labeled probesof DNA sequences, one can isolate homologous or related genes.

[0086] Identification of Novel Promoter Elements

[0087] The sequence of the 5′ flanking region may be utilized forpromoter elements, including enhancer binding sites, that provide forregulation in tissues where the subject nucleic acids are expressed. Thetissue specific expression is useful for determining the pattern ofexpression, and for providing promoters that mimic the native pattern ofexpression. Naturally occurring polymorphisms in the promoter region areuseful for determining natural variations in expression, particularlythose that may be associated with disease.

[0088] Identification of Naturally-occurring Factors RegulatingExpression

[0089] Alternatively, mutations may be introduced into the promoterregion to determine the effect of altering expression in experimentallydefined systems. Methods for the identification of specific DNA motifsinvolved in the binding of transcriptional factors are known in the art,e.g., sequence similarity to known binding motifs, gel retardationstudies, etc. For examples, see Blackwell et al. (1995), Mol. Med.1:194-205; Mortlock et al. (1996), Genome Res. 6:327-33; and Joulin andRichard-Foy (1995), Eur. J. Biochem. 232:620-626.

[0090] The regulatory sequences may be used to identify cis actingsequences required for transcriptional or translational regulation ofnucleic acid expression, especially in different tissues or stages ofdevelopment, and to identify cis acting sequences and trans-actingfactors that regulate or mediate nucleic acid expression. Suchtranscription or translational control regions may be operably linked toan a polynucleotide of the invention in order to promote expression incultured cells, or in embryonic, fetal or adult tissues, and for genetherapy.

[0091] Probes and Primers

[0092] The polynucleotides of the invention can also be used in thedesign of primers or probes. Small DNA fragments (e.g., less than 100nt) are useful as probes or primers, as in PCR, hybridization screening,etc. Larger DNA fragments, e.g., greater than 100 nt, are also usefulfor production of the encoded polypeptide, as described above. For usein amplification reactions, such as PCR, a pair of primers will be used.The exact composition of the primer sequences is not critical to theinvention, but for most applications the primers will hybridize to thesubject sequence under stringent conditions, particularly underconditions of high stringency, as known in the art. The pair of primersare usually chosen so as to generate an amplification product of atleast about 50 nt, more usually at least about 100 nt. Algorithms forthe selection of primer sequences are generally known, and are availablein commercial software packages.

[0093] Identification of Expression Patterns in Biological Specimens

[0094] The polynucleotides of the invention may also be used to identifyexpression of the gene in a biological specimen. Detection of expressionof a particular nucleotide sequence in a selected cell is wellestablished in the literature. Briefly, mRNA is isolated from a cellsample. The mRNA may be amplified by RT-PCR, using reverse transcriptaseto form a complementary DNA (cDNA) strand, followed by polymerase chainreaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g.,nitrocellulose, nylon, etc., and then probed with a fragment of thesubject DNA as a probe. Other techniques, such as oligonucleotideligation assays, in situ hybridizations, and hybridization tooligonucleotides arrayed on a solid chip may also find use. Detection ofmRNA hybridizing to, or amplified by primers specific for, the subjectsequence is indicative of expression of a differentially expressed genein the sample. This particular use of the polynucleotides of theinvention is described below in further detail.

[0095] Preparation of Mutants

[0096] The sequence of a differentially expressed gene, includingflanking promoter regions and coding regions, may be mutated in variousways known in the art to generate targeted changes in promoter strength,sequence of the encoded protein, etc. The DNA sequence or proteinproduct of such mutants will usually be substantially similar to thesequences provided herein, e.g., will differ by at least one nucleotideor amino acid, and may differ by at least two, up to ten or morenucleotides or amino acids. The sequence changes may be substitutions(conservative or non-conservative), insertions, deletions, or acombination thereof. Deletions may further include larger changes, suchas deletions of a domain or exon. Other modifications of interestinclude additions, such as in epitope tagging, e.g., with the FLAGsystem, HA, green fluorescent proteins (GFP), etc.

[0097] Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for site specific mutagenesis may be found inGustin et al. (1993), Biotechniques 14:22; Barany (1985), Gene37:111-23; Colicelli et al., (1985), Mol. Gen. Genet. 199:537-9; andPrentki et al. (1984), Gene 29:303-13. Methods for site specificmutagenesis can be found in Sambrook et al., Molecular Cloning: ALaboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al.(1993), Gene 126:35-41; Sayers et al. (1992), Biotechniques 13:592-6;Jones and Winistorfer (1992), Biotechniques 12:528-30; Barton et al.(1990), Nucleic Acids Res 18:7349-55; Marotti and Tomich (1989), GeneAnal. Tech. 6:67-70; and Zhu (1989), Anal Biochem 177:120-4. Suchmutated genes may be used to study structure-function relationships ofdifferentially expressed polypeptides, or to alter properties of theprotein that affect its function or regulation (e.g., to providepolynucleotides or polypeptides enhanced or diminished in a selectedactivity).

[0098] Production of in Vivo Models of Differentially ExpressedPolynucleotide Function

[0099] The subject nucleic acids can be used to generate transgenic,non-human animals or site specific gene modifications in cell lines.“Transgenic animals” encompasses genetically modified, non-human hostshaving a deletion (“knock-out”) of one or both alleles of adifferentially expressed gene, or an introduced copy (“knock-in”) of anendogenous or exogenous differentially expressed gene. “Transgenicanimals” also encompasses conditional knock-outs and other transgenicanimals altered for expression of a polynucleotide of the invention.Transgenic animals may be made through homologous recombination, wherethe normal locus of the particular gene of interest is altered, or maybe made by random genomic integration or episomal maintenance of anucleic acid construct into the host genome. The host animal may be ofany suitable genus or species, particularly a mammal (e.g., rodents(mice, rats, etc.), cows, pigs, goats, horses, etc.

[0100] The modified cells or animals are useful in the study ofdifferentially expressed gene function and regulation. For example, aseries of small deletions and/or substitutions may be made in the host'snative gene to determine the role of different exons in differentiallyexpressed gene activity. Specific constructs of interest includeanti-sense nucleic acid compositions, which will block gene expression,expression of dominant negative mutations, and over-expression of genes.Where a particular sequence is introduced, the introduced sequence maybe either a complete or partial sequence of a gene native to the host,or may be a complete or partial sequence that is exogenous to the hostanimal, e.g., a human sequence. A detectable marker, such as lac Z, maybe introduced into the locus, where upregulation of gene expression willresult in an easily detected change in phenotype. One may also providefor expression of the differentially expressed gene or variants thereofin cells or tissues where it is not normally expressed, at levels notnormally present in such cells or tissues, or at abnormal times ofdevelopment.

[0101] Vectors for use in production of transgenic animals are known inthe art (e.g., plasmids, retroviral vectors (as wells as vectors basedon other animal viruses), YACs, etc. Conveniently, markers for positiveand negative selection are included. Methods for generating cells havingtargeted gene modifications through homologous recombination are knownin the art. For various techniques for transfecting mammalian cells, seeKeown et al. (1990), Meth. Enzymol. 185:527-537.

[0102] Transgenic animals may be produced according to methods wellknown in the art. For example, transgenic animals may be produced usingembryonic stem (ES) cells. An ES cell line may be employed, or embryoniccells may be obtained freshly from a host, e.g., mouse, rat, guinea pig,etc. Such cells are grown on an appropriate fibroblast-feeder layer orgrown in the presence of leukemia inhibiting factor (LIF). When ES orembryonic cells have been transformed, they may be used to producetransgenic animals. After transformation, the cells are plated onto afeeder layer in an appropriate medium. Cells containing the constructmay be detected by employing a selective medium. After sufficient timefor colonies to grow, they are picked and analyzed for the occurrence ofhomologous recombination or integration of the construct. Those coloniesthat are positive may then be used for embryo manipulation andblastocyst injection. Blastocysts are obtained from 4 to 6 week oldsuperovulated females. The ES cells are trypsinized, and the modifiedcells are injected into the blastocoel of the blastocyst. Afterinjection, the blastocysts are returned to each uterine horn ofpseudopregnant females. Females are then allowed to go to term and theresulting offspring screened for the construct. By providing for adifferent phenotype of the blastocyst and the genetically modifiedcells, chimeric progeny can be readily detected.

[0103] The chimeric animals are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogeneic or congenic grafts or transplants, or in in vitro culture.The transgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc., e.g., to determine the effectof a candidate drug on activity of the gene product of thedifferentially expressed gene.

[0104] Production of in Vitro Models of Function of DifferentiallyExpressed Genes

[0105] One can also use the compositions of the subject invention toproduce in vitro models of differentially expressed gene function, wherethe format of such models can be readily determined by those of skill inthe art. For example, the differentially expressed polynucleotides ofthe invention can be used in conjunction with in vitro cell lines toexamine the effect of modification of expression (e.g., overexpressionor inhibition of expression) of a selected polynucleotide of theinvention. For example where a differentially expressed polynucleotideis substantially selectively underexpressed in a cancerous pancreaticcell, the in vitro expression system can use an appropriate cancerouspancreatic cell line to examine the effect of increasing expression ofthe polynucleotide. Where increasing expression the underexpressedpolynucleotide in the cancerous cell line results in a desirable effect(e.g., inhibition of growth of the cell line, affect upon morphology,etc.), the differentially expressed polynucleotide is identified ascorresponding to a gene that plays an important role in development orregulation of the cancerous phenotype, and thus may be useful as atherapeutic agent or target of a therapeutic agent.

[0106] Libraries and Computer-related Embodiments

[0107] A library of polynucleotides is a collection of sequenceinformation, which information is provided in either biochemical form(e.g., as a collection of polynucleotide molecules), or in electronicform (e.g., as a collection of polynucleotide sequences stored in acomputer-readable form, as in a computer system and/or as part of acomputer program), where the sequence information of the polynucleotidesserve as markers of a particular pancreatic disease. A marker of apancreatic disease is a representation of a cellular product that ispresent either at an increased or decreased level relative to normalpancreatic cells. For example, a polynucleotide sequence in a librarymay be a polynucleotide that represents an mRNA, polypeptide, or othergene product encoded by the polynucleotide, that is either overexpressedor underexpressed in a cell affected by pancreatic disease relative to anormal (i.e., substantially disease-free) pancreatic cell.

[0108] The nucleotide sequence information of the library may beembodied in any suitable form, e.g., electronic or biochemical forms.For example, a library of sequence information embodied in electronicform includes an accessible computer data file that contains therepresentative nucleotide sequences of genes that are differentiallyexpressed (e.g., overexpressed or underexpressed) as between, forexample, i) a cancerous pancreatic cell and a normal pancreatic cell;ii) a cancerous pancreatic cell and a dysplastic pancreatic cell; iii) acancerous pancreatic cell and a pancreatic cell affected bypancreatitis; iv) a pancreatic cell of an individual affected bydiabetes (Type I or Type II) and a normal pancreatic cell (e.g.,unaffected by diabetes); and/or v) a dysplastic pancreatic cell relativeto a normal pancreatic cell. Other combinations and comparisons ofpancreatic cells of various disease will be readily apparent to theordinarily skilled artisan. Biochemical embodiments of the libraryinclude a collection of nucleic acids that have the sequences of thegenes in the library, where the nucleic acids may correspond to theentire gene in the library or to a fragment thereof, as described ingreater detail below.

[0109] The differentially expressed polynucleotides of the library havenucleotide sequences of at least about 10 nt to about 100 nt, usually atleast about 20 nt to 200 nt, and more usually at least about 50 nt toabout 500 nt or more, up to about 300 nt to about 1,000 nt to about1,500 nt of genes that are differentially expressed in pancreatic cellsthat are differentially affected by a disease or condition (e.g.,cancer, dysplasia, pancreatitis, diabetes, normal, etc.). Thepolynucleotide libraries of the subject invention include sequenceinformation of a plurality of polynucleotide sequences, where at leastone of the polynucleotides has a sequence of any of SEQ ID NOS: 1-6. Byplurality is meant at least 2, usually at least 3 and may include allsix of SEQ ID NOS: 1-6. The length and number of polynucleotides in thelibrary will vary with the nature of the library, e.g., if the libraryis an oligonucleotide array, a cDNA array, a computer database of thesequence information, etc.

[0110] Where the library is an electronic library, the nucleic acidsequence information can be present in a variety of media. “Media”refers to a manufacture, other than an isolated nucleic acid molecule,that contains the sequence information of the present invention. Such amanufacture provides the genome sequence or a subset thereof in a formthat can be examined by means not directly applicable to the sequence asit exists in a nucleic acid. For example, the nucleotide sequence of thepresent invention, e.g. the nucleic acid sequences of any of thepolynucleotides of SEQ ID NOS: 1-6, can be recorded on computer readablemedia, e.g. any medium that can be read and accessed directly by acomputer. Such media include, but are not limited to: magnetic storagemedia, such as a floppy disc, a hard disc storage medium, and a magnetictape; optical storage media such as CD-ROM; electrical storage mediasuch as RAM and ROM; and hybrids of these categories such asmagnetic/optical storage media. One of skill in the art can readilyappreciate how any of the presently known computer readable mediums canbe used to create a manufacture comprising a recording of the presentsequence information. “Recorded” refers to a process for storinginformation on computer readable medium, using any such methods as knownin the art. Any convenient data storage structure may be chosen, basedon the means used to access the stored information. A variety of dataprocessor programs and formats can be used for storage, e.g. wordprocessing text file, database format, etc.

[0111] By providing the nucleotide sequence in computer readable form,the information can be accessed for a variety of purposes. Computersoftware to access sequence information is publicly available. Forexample the BLAST (Altschul et al., supra.) and BLAZE (Brutlag etal.(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase systemcan be used identify open reading frames (ORFs) within the genome thatcontain homology to ORFs or proteins from other organisms.

[0112] As used herein, “a computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thenucleotide sequence information of the present invention. The minimumhardware of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. A skilled artisan can readily appreciate thatany one of the currently available computer-based system are suitablefor use in the present invention. The data storage means may compriseany manufacture comprising a recording of the present sequenceinformation as described above, or a memory access means that can accesssuch a manufacture.

[0113] “Search means” refers to one or more programs implemented on thecomputer-based system, to compare a target sequence or target structuralmotif with the stored sequence information. Search means are used toidentify fragments or regions of the genome that match a particulartarget sequence or target motif. A variety of known algorithms arepublicly known and commercially available, e.g. MacPattern (EMBL),BLASTN and BLASTX (NCBI). A “target sequence” can be any DNA or aminoacid sequence of six or more nucleotides or two or more amino acids,preferably from about 10 to 100 amino acids or from about 30 to 300nucleotide residues.

[0114] A “target structural motif,” or “target motif,” refers to anyrationally selected sequence is or combination of sequences in which thesequence(s) are chosen based on a three-dimensional configuration thatis formed upon the folding of the target motif, or on consensussequences of regulatory or active sites. There are a variety of targetmotifs known in the art. Protein target motifs include, but arc notlimited to, enzyme active sites and signal sequences. Nucleic acidtarget motifs include, but are not limited to, hairpin structures,promoter sequences and other expression elements such as binding sitesfor transcription factors.

[0115] A variety of structural formats for the input and output meanscan be used to input and output the information in the computer-basedsystems of the present invention. One format for an output means ranksfragments of the genome possessing varying degrees of homology to atarget sequence or target motif. Such presentation provides a skilledartisan with a ranking of sequences and identifies the degree ofsequence similarity contained in the identified fragment.

[0116] A variety of comparing means can be used to compare a targetsequence or target motif with the data storage means to identifysequence fragments of the genome. A skilled artisan can readilyrecognize that any one of the publicly available homology searchprograms can be used as the search means for the computer based systemsof the present invention.

[0117] As discussed above, the “library” of the invention alsoencompasses biochemical libraries of the differentially expressedpolynucleotides of SEQ ID NOS: 1-6, e.g., collections of nucleic acidsrepresenting these differentially expressed sequences. The biochemicallibraries may take a variety of forms, e.g. a solution of cDNAs, apattern of probe nucleic acids stably associated with a surface of asolid support, i.e. an array, and the like. Of particular interest arenucleic acid arrays in which one or more of SEQ ID NOS: 1-6 isrepresented on the array. By array is meant a an article of manufacturethat has at least a substrate with at least two distinct nucleic acidtargets on one of its surfaces, where the number of distinct nucleicacids may be considerably higher, typically being at least 10 nt,usually at least 20 nt and often at least 25 nt. A variety of differentarray formats have been developed and are known to those of skill in theart, including those described in U.S. Pat. Nos. 5,242,974; 5,384,261;5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,445,934;5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,556,752;5,561,071; 5,599,895; 5,624,711; 5,639,603; 5,658,734; WO 93/17126; WO95/11995; WO 95/35505; EP 742 287; and EP 799 897, the disclosures ofwhich are herein incorporated by reference. The arrays of the subjectinvention find use in a variety of applications, including geneexpression analysis, drug screening, mutation analysis and the like, asdisclosed in the above listed patent documents.

[0118] In addition to the above nucleic acid libraries, analogouslibraries of differentially expressed polypeptides are also provided,where the where the polypeptides of the library will represent at leasta portion of the polypeptides encoded by SEQ ID NOS: 1-6.

[0119] Diagnostic Applications

[0120] Also provided are methods of diagnosing disease states associatedwith expression of differentially expressed genes, e.g., based onobserved levels of a differentially expressed polypeptide or theexpression level of a differentially expressed gene in a biologicalsample of interest. In general, the diagnostic methods of the inventioninvolve detection of a level or amount of a differentially expressedgene product in a test sample obtained from a patient suspected ofhaving or being susceptible to a pancreatic disease (e.g., cancer,pancreatitis, diabetes, etc.), and comparing the detected levels tothose levels found in normal cells (e.g., cells substantially unaffectedby cancer) and/or other control cells (e.g., to differentiate acancerous cell from a cell affected by dysplasia or pancreatitis).Furthermore, the severity of the disease may be assessed by comparingthe detected levels of differentially expressed gene product with thoselevels detected in samples representing the levels of differentiallygene product associated with varying degrees of severity of pancreaticcancer.

[0121] Diagnostic methods of the subject invention typically involvecomparison of the abundance of a selected differentially expressed geneproduct in a sample of interest with that of a control to determine anyrelative differences in the expression of the gene product, where thedifference may be measured qualitatively and/or quantitatively. Thedifferences in expression are then correlated with the presence orabsence of an abnormal expression pattern. A variety of differentmethods for determining the nucleic acid abundance in a sample are knownto those of skill in the art, where particular methods of interestinclude those described in: Pietu et al. (1996) Genome Res. 6:492; Zhaoet al. (1995) Gene 156:207; Soares, (1977) Curr. Opin. Biotechnol. 8:542-546; Raval, (1994) J. Pharmacol Toxicol Methods 32:125; Chalifour etal. (1994) Anal. Biochem 216:299; Stolz et al., (1996) Mol. Biotechnol.6:225; Hong et al., (1982) Biosci. Reports 2:907; and McGraw, (1984)Anal. Biochem. 143:298. Also of interest are the methods disclosed in WO97/27317, the disclosure of which is herein incorporated by reference.

[0122] In general, diagnostic assays of the invention involve detectionof a gene product of a the polynucleotide sequence (e.g., mRNA orpolypeptide) that corresponds to a sequence set forth in any one of SEQID NOS: 1-6. The patient from whom the sample is obtained can beapparently healthy, susceptible to pancreatic disease (e.g., asdetermined by family history or exposure to certain environmentalfactors), or can already be identified as having a condition in whichaltered expression of a gene product of the invention is implicated.

[0123] The level of a particular expression product of a polynucleotidesequence of the invention in a sample can be determined qualitatively orquantitatively. Quantitation can be accomplished, for example, bycomparing the level of expression product detected in the sample withthe amounts of product present in a standard curve. A comparison can bemade visually or using a technique such as densitometry, with or withoutcomputerized assistance.

[0124] In the assays of the invention, the diagnosis may be determinedbased on detected gene product expression levels of a gene productencoded by at least one, preferably at least two or more, at least 3 ormore, or at least 4 or more of the polynucleotides having a sequence setforth in SEQ ID NOS: 1-6, and may involve detection of expression ofgenes corresponding to all 6 of SEQ ID NOS: 1-6 and/or additionalsequences that can serve as additional diagnostic markers and/orreference sequences. Where the diagnostic method is designed to detectthe presence or susceptibility of a patient to pancreatic cancer, theassay preferably involves detection of a gene product encoded by apolynucleotide having the sequence of SEQ ID NO: 2, which isoverexpressed in pancreatic cancer. Where the diagnostic method isdesigned to detect the presence or susceptibility of a patient topancreatic dysplasia, the assay preferably involves detection of atleast one of the gene products encoded by a polynucleotide having thesequence of SEQ ID NOS: 4 and 5, which are overexpressed in pancreaticdysplasia, and/or SEQ ID NOS: 1 and 3, which are underexpressed inpancreatic dysplasia. Diagnosis of pancreatic cancer and/or pancreaticdysplasia can also involve the detection of the gene product encoded bya polynucleotide having the sequence set forth in SEQ ID NO: 6, which isunderexpressed in both pancreatic cancer and dysplasia.

[0125] Any of a variety of detectable labels can be used in connectionwith the various embodiments of the diagnostic methods of the invention.Suitable detectable labels include fluorochromes,(e.g. fluoresceinisothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,allophycocyanin, 6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA)),radioactive labels, (e.g. ³²P, ³⁵S, ³H, etc.), and the like. Thedetectable label may involve a two stage systems (e.g., biotin-avidin,hapten-anti-hapten antibody, etc.)

[0126] Reagents specific for the polynucleotides and polypeptides of theinvention, such as antibodies and nucleotide probes, can be supplied ina kit for detecting the presence of an expression product in abiological sample. The kit can also contain buffers or labelingcomponents, as well as instructions for using the reagents to detect andquantify expression products in the biological sample. Exemplaryembodiments of the diagnostic methods of the invention are describedbelow in more detail.

[0127] Polypeptide Detection in Diagnosis

[0128] In one embodiment, the test sample is assayed for the level of adifferentially expressed polypeptide. Diagnosis may be accomplishedusing any of a number of methods to determine the absence or presence oraltered amounts of the differentially expressed polypeptide in the testsample. For example, detection may utilize staining of cells orhistological sections with labeled antibodies, performed in accordancewith conventional methods. Cells may be permeabilized to staincytoplasmic molecules. In general, antibodies that specifically bind adifferentially expressed polypeptide of the invention are added to asample, and incubated for a period of time sufficient to allow bindingto the epitope, usually at least about 10 minutes. The antibody may bedetectably labeled for direct detection (e.g., using radioisotopes,enzymes, fluorescers, chemiluminescers, and the like), or may be used inconjunction with a second stage antibody or reagent to detect binding(e.g., biotin with horseradish peroxidase-conjugated avidin, a secondaryantibody conjugated to a fluorescent compound, e.g. fluorescein,rhodamine, Texas red, etc.). The absence or presence of antibody bindingmay be determined by various methods, including flow cytometry ofdissociated cells, microscopy, radiography, scintillation counting, etc.Any suitable alternative methods can of qualitative or quantitativedetection of levels or amounts of differentially expressed polypeptidemay be used, for example ELISA, western blot, immunoprecipitation,radioimmunoassay, etc.

[0129] In general, the detected level of differentially expressedpolypeptide in the test sample is compared to a level of thedifferentially expressed gene product in a reference or control sample,e.g., in a normal cell (negative control) or in a cell having a knowndisease state (positive control). For example, a higher level ofexpression of a polypeptide encoded by SEQ ID NO: 2 relative to a levelassociated with a normal sample is indicative of the presence ofpancreatic cancer in the patient from whom the sample is derived. Ahigher level of expression of a polypeptide encoded by SEQ ID NOS: 4 or5 relative to a level associated with a normal sample is indicative ofthe presence of pancreatic dysplasia, while detection of a lower levelof expression of a polypeptide encoded by SEQ ID NOS: 1 or 3 relative toa level associated with a normal sample is indicative of the presence ofpancreatic dysplasia in the patient from whom the sample is derived.Detection of a lower level of the polypeptide encoded by SEQ ID NO: 6relative to a normal level is indicative of the presence of pancreaticcancer and/or dysplasia in the patient.

[0130] mRNA Detection

[0131] The diagnostic methods of the invention can also or alternativelyinvolve detection of mRNA encoded by a gene corresponding to adifferentially expressed polynucleotides of the invention. Any suitablequalitative or quantitative methods known in the art for detectingspecific mRNAs can be used. mRNA can be detected by, for example, insitu hybridization in tissue sections, by reverse transcriptase-PCR, orin Northern blots containing poly A+ mRNA. One of skill in the art canreadily use these methods to determine differences in the size or amountof mRNA transcripts between two samples. For example, the level of mRNAof the invention in a tissue sample suspected of being cancerous ordysplastic is compared with the expression of the mRNA in a referencesample, e.g., a positive or negative control sample (e.g., normaltissue, cancerous tissue, etc.).

[0132] In general, detection in the test sample of a higher level ofmRNA expressed from a polynucleotide sequence having a sequence of SEQID NO: 2, as compared to the normal tissue, indicates the presence ofpancreatic cancerous cells in the suspect tissue. A higher level of mRNAhaving a sequence corresponding to a sequence of SEQ ID NOS: 4 or 5, ascompared to the normal tissue, indicates the presence dysplastic cellsin the test sample. A lower level of the mRNA having a sequencecorresponding to a sequence of SEQ ID NOS: 1 or 3, as compared to thenormal tissue, indicates the presence of dysplastic cells in the testsample. A lower level of mRNA having a sequence corresponding to asequence of SEQ ID NO: 6 is indicative of the presence of cancerousand/or dysplastic cells in the test sample. Any combinations of thesesequences can be used in diagnosis.

[0133] Any suitable method for detecting and comparing mRNA expressionlevels in a sample can be used in connection with the diagnostic methodsof the invention (see, e.g., U.S. Pat. No. 5,804,382). For example, mRNAexpression levels in a sample can be determined by generation of alibrary of expressed sequence tags (ESTs) from the sample, where the ESTlibrary is representative of sequences present in the sample (Adams, etal., (1991) Science 252:1651). Enumeration of the relativerepresentation of ESTs within the library can be used to approximate therelative representation of the gene transcript within the startingsample. The results of EST analysis of a test sample can then becompared to EST analysis of a reference sample to determine the relativeexpression levels of a selected polynucleotide, particularly apolynucleotide corresponding to one or more of the differentiallyexpressed genes described herein.

[0134] Alternatively, gene expression in a test sample can be performedusing serial analysis of gene expression (SAGE) methodology (Velculescuet al. (1995) Science 270:484). In short, SAGE involves the isolation ofshort unique sequence tags from a specific location within eachtranscript (e.g., a sequence of any one of SEQ ID NOS: 1-6). Thesequence tags are concatenated, cloned, and sequenced. The frequency ofparticular transcripts within the starting sample is reflected by thenumber of times the associated sequence tag is encountered with thesequence population.

[0135] Gene expression in a test sample can also be analyzed usingdifferential display (DD) methodology. In DD, fragments defined byspecific sequence delimiters (e.g., restriction enzyme sites) are usedas unique identifiers of genes, coupled with information about fragmentlength or fragment location within the expressed gene. The relativerepresentation of an expressed gene with a sample can then be estimatedbased on the relative representation of the fragment associated withthat gene within the pool of all possible fragments. Methods andcompositions for carrying out DD are well known in the art, see, e.g.,U.S. Pat. No. 5,776,683; and U.S. Pat. No. 5,807,680.

[0136] Alternatively, gene expression in a sample using hybridizationanalysis, which is based on the specificity of nucleotide interactions.Oligonucleotides or cDNA can be used to selectively identify or captureDNA or RNA of specific sequence composition, and the amount of RNA orcDNA hybridized to a known capture sequence determined qualitatively orquantitatively, to provide information about the relative representationof a particular message within the pool of cellular messages in asample. Hybridization analysis can be designed to allow for concurrentscreening of the relative expression of hundreds to thousands of genesby using, for example, array-based technologies having high densityformats, including filters, microscope slides, or microchips, orsolution-based technologies that use spectroscopic analysis (e.g., massspectrometry). One exemplary use of arrays in the diagnostic methods ofthe invention is described below in more detail.

[0137] Use of a Single Gene in Diagnostic Applications

[0138] The diagnostic methods of the invention may also focus on theexpression of a single differentially expressed gene. For example, thediagnostic method may involve detecting a differentially expressed gene,or a polymorphism of such a gene (e.g., a polymorphism in an codingregion or control region), that is associated with disease.Disease-associated polymorphisms may include deletion or truncation ofthe gene, mutations that alter expression level and/or affect activityof the encoded protein, etc.

[0139] Changes in the promoter or enhancer sequence that may affectexpression levels of an differentially gene can be compared toexpression levels of the normal allele by various methods known in theart. Methods for determining promoter or enhancer strength includequantitation of the expressed natural protein; insertion of the variantcontrol element into a vector with a reporter gene such asβ-galactosidase, luciferase, chloramphenicol acetyltransferase, etc.that provides for convenient quantitation; and the like.

[0140] A number of methods are available for analyzing nucleic acids forthe presence of a specific sequence, e.g. a disease associatedpolymorphism. Where large amounts of DNA are available, genomic DNA isused directly. Alternatively, the region of interest is cloned into asuitable vector and grown in sufficient quantity for analysis. Cellsthat express a differentially expressed gene may be used as a source ofmRNA, which may be assayed directly or reverse transcribed into cDNA foranalysis. The nucleic acid may be amplified by conventional techniques,such as the polymerase chain reaction (PCR), to provide sufficientamounts for analysis, and a detectable label may be included in theamplification reaction (e.g., using a detectably labeled primer ordetectably labeled oligonucleotides) to facilitate detection. The use ofthe polymerase chain reaction is described in Saiki, et al. (1985),Science 239:487, and a review of techniques may be found in Sambrook, etal. (1989), Molecular Cloning: A Laboratory Manual, pp. 14.2-14.33.Alternatively, various methods are known in the art that utilizeoligonucleotide ligation as a means of detecting polymorphisms, forexamples see Riley et al. (1990), Nucl. Acids Res. 18:2887-2890; andDelahunty et al. (1996), Am. J. Hum. Genet. 58:1239-1246.

[0141] The sample nucleic acid, e.g. amplified or cloned fragment, isanalyzed by one of a number of methods known in the art. The nucleicacid may be sequenced by dideoxy or other methods, and the sequence ofbases compared to a selected sequence, e.g., to a wild-type sequence.Hybridization with the polymorphic or variant sequence may also be usedto determine its presence in a sample (e.g., by Southern blot, dot blot,etc.). The hybridization pattern of a polymorphic or variant sequenceand a control sequence to an array of oligonucleotide probes immobilizedon a solid support, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be used as a means of identifying polymorphic orvariant sequences associated with disease. Single strand conformationalpolymorphism (SSCP) analysis, denaturing gradient gel electrophoresis(DGGE), and heteroduplex analysis in gel matrices are used to detectconformational changes created by DNA sequence variation as alterationsin electrophoretic mobility. Alternatively, where a polymorphism createsor destroys a recognition site for a restriction endonuclease, thesample is digested with that endonuclease, and the products sizefractionated to determine whether the fragment was digested.Fractionation is performed by gel or capillary electrophoresis,particularly acrylamide or agarose gels.

[0142] Screening for mutations in an differentially expressed gene maybe based on the functional or antigenic characteristics of the protein.Protein truncation assays are useful in detecting deletions that mayaffect the biological activity of the protein. Various immunoassaysdesigned to detect polymorphisms in proteins may be used in screening.Where many diverse genetic mutations lead to a particular diseasephenotype, functional protein assays have proven to be effectivescreening tools. The activity of the encoded protein may be determinedby comparison with the wild-type protein.

[0143] Pattern Matching in Diagnosis Using Arrays

[0144] In another embodiment, the diagnostic methods of the inventioninvolve detection of expression of a selected set of genes in a testsample to produce a test expression pattern (TEP). The TEP is comparedto a reference expression pattern (REP), which is generated by detectionof expression of the selected set of genes in a reference sample (e.g.,a positive or negative control sample). The selected set of genesincludes at least one of the differentially expressed genes of theinvention, which genes correspond to the polynucleotide sequences of SEQID NOS: 1-6.

[0145] REPs can be generated in a variety of ways according to methodswell known in the art. For example, REPs can be generated by hybridizinga control sample to an array having a selected set of polynucleotides(particularly a selected set of differentially expressedpolynucleotides), acquiring the hybridization data from the array, andstoring the data in a format that allows for ready comparison of the REPwith a TEP. Alternatively, all expressed sequences in a control samplecan be isolated and sequenced, e.g., by isolating mRNA from a controlsample, converting the mRNA into cDNA, and sequencing the cDNA. Theresulting sequence information roughly or precisely reflects theidentity and relative number of expressed sequences in the sample. Thesequence information can then be stored in a format (e.g., acomputer-readable format) that allows for ready comparison of the REPwith a TEP. The REP can be normalized prior to or after data storage,and/or may be processed to selectively remove sequences of expressedgenes that are of less interest or that may complicate analysis (e.g.,some or all of the sequences associated with housekeeping genes may beeliminated from the REP data).

[0146] TEPs can be generated in a manner similar to REPs, e.g., byhybridizing a test sample to an array having a selected set ofpolynucleotides, particularly a selected set of differentially expressedpolynucleotides, acquiring the hybridization data from the array, andstoring the data in a format that allows for ready comparison of the TEPwith a REP. The REP and TEP to be used in a comparison may be generatedsimultaneously, or the TEP may be compared to previously generated andstored REPs.

[0147] In one embodiment of the invention, comparison of a TEP with aREP involves hybridizing a test sample with a reference array, where thereference array has one or more reference sequences for use inhybridization with a sample. The reference sequences include all, atleast one of, or any subset of the differentially expressedpolynucleotides listed in Table 1. Hybridization data for the testsample is acquired, the data normalized, and the produced TEP comparedwith a REP generated using an array having the same or similar selectedset of differentially expressed polynucleotides. Probes that correspondto sequences differentially expressed between the two samples will showdecreased or increased hybridization efficiency for one of the samplesrelative to the other.

[0148] Reference arrays may be produced according to any suitablemethods known in the art. For example, methods of producing large arraysof oligonucleotides are described in U.S. Pat. No. 5,134,854, and U.S.Pat. No. 5,445,934 using light-directed synthesis techniques. Using acomputer controlled system, a heterogeneous array of monomers isconverted, through simultaneous coupling at a number of reaction sites,into a heterogeneous array of polymers. Alternatively, microarrays aregenerated by deposition of pre-synthesized oligonucleotides onto a solidsubstrate, for example as described in PCT published application no. WO95/35505.

[0149] Methods for collection of data from hybridization of samples witha reference arrays are also well known in the art. For example, thepolynucleotides of the reference and test samples can be generated usinga detectable fluorescent label, and hybridization of the polynucleotidesin the samples detected by scanning the microarrays for the presence ofthe detectable label. Methods and devices for detecting fluorescentlymarked targets on devices are known in the art. Generally, suchdetection devices include a microscope and light source for directinglight at a substrate. A photon counter detects fluorescence from thesubstrate, while an x-y translation stage varies the location of thesubstrate. A confocal detection device that may be used in the subjectmethods is described in U.S. Pat. No. 5,631,734. A scanning lasermicroscope is described in Shalon et al. (1996) Genome Res. 6:639. Ascan, using the appropriate excitation line, is performed for eachfluorophore used. The digital images generated from the scan are thencombined for subsequent analysis. For any particular array element, theratio of the fluorescent signal from one sample (e.g., a test sample) iscompared to the fluorescent signal from another sample (e.g., areference sample), and the relative signal intensity determined.

[0150] Methods for analyzing the data collected from hybridization toarrays are well known in the art. For example, where detection ofhybridization involves a fluorescent label, data analysis may includethe steps of determining fluorescent intensity as a function ofsubstrate position from the data collected, removing outliers, i.e. datadeviating from a predetermined statistical distribution, and calculatingthe relative binding affinity of the targets from the remaining data.The resulting data may be displayed as an image with the intensity ineach region varying according to the binding affinity between targetsand probes.

[0151] In general, the test sample is classified as having a geneexpression profile corresponding to that associated with a disease ornon-disease state (e.g., pancreatic cancer, pancreatic dysplasia,pancreatitis, diabetes, normal, etc.) by comparing the TEP generatedfrom the test sample to one or more REPs generated from referencesamples (e.g., from samples associated with pancreatic cancer,pancreatic dysplasia, pancreatitis, diabetes, normal, etc.). Thecriteria for a match or a substantial match between a TEP and a REPinclude expression of the same or substantially the same set ofreference genes, as well as expression of these reference genes atsubstantially the same levels (e.g., no significant difference betweenthe samples for a signal associated with a selected reference sequenceafter normalization of the samples, or at least no greater than about25% to about 40% difference in signal strength for a given referencesequence. In general, a pattern match between a TEP and a REP includes amatch in expression, preferably a match in qualitative or quantitativeexpression level, of at least one of, all or any subset of thedifferentially expressed genes of the invention as represented by SEQ IDNOS: 1-6.

[0152] Pattern matching may be performed manually, or may be performedusing a computer program. Methods for preparation of substrate matrices(e.g., arrays), design of oligonucleotides for use with such matrices,labeling of probes, hybridization conditions, scanning of hybridizedmatrices, and analysis of patterns generated, including comparisonanalysis, are described in, for example, U.S. Pat. No. 5,800,992.

[0153] Screening Assays

[0154] The differentially expressed polynucleotides and polypeptides ofthe invention can be used in a screening assay designed to identifyagents that modulate activity of the differentially expressed geneproduct, e.g., by modulating expression (e.g., enhancing or inhibitingexpression), by modulating polypeptide activity (e.g., enhancing orinhibiting a biological activity), and the like. The screening methodswill typically be assays that provide for qualitative and/orquantitative measurements of biological activity in the presence of aparticular candidate therapeutic agent. For example, the assay maymeasure activity a polypeptide in the presence and absence of acandidate inhibitor agent, or may examine the effect of a candidateagent upon expression of a selected polynucleotide.

[0155] The screening method may be an in vitro or in vivo format, whereboth formats are readily developed by those of skill in the art.Depending on the particular method, one or more of, usually one of, thecomponents of the screening assay may be detectably labeled, e.g. usinga fluorescent or radioactive tag, or a member of a multicomponent signalproducing system, e.g. biotin for binding to an enzyme-streptavidinconjugate in which the enzyme is capable of converting a substrate to achromogenic product. A variety of other reagents may be included in thescreening assay. These include reagents like salts, neutral proteins,e.g. albumin, detergents, etc that are used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used.

[0156] A variety of different candidate agents may be screened in suchscreening assays. Candidate agents encompass numerous chemical classes,though typically they are organic molecules, preferably small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

[0157] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0158] Drug screening may be performed using an in vitro model, agenetically altered cell or animal, or purified differentially expressedpolypeptide to facilitate identification of ligands or substrates thatbind to, modulate or mimic the action of a differentially expressedpolypeptide. Areas of particular interest include the development ofcancer treatments, metastasis, etc. Drug screening may also be performedfor identification of agents that provide a replacement for or increasethe function of a differentially expressed polypeptide that isunderexpressed in abnormal cells. Conversely, agents that inhibitfunction of a differentially expressed polypeptide that is overexpressedin abnormal cells are predicted to inhibit the process of disease (e.g.oncogenesis). Of particular interest are screening assays for agentsthat have a low toxicity for human cells. A wide variety of assays maybe used for this purpose, including labeled in vitro protein-proteinbinding assays, electrophoretic mobility shift assays, immunoassays forprotein binding, and the like. The purified protein may also be used fordetermination of three-dimensional crystal structure, which can be usedfor modeling intermolecular interactions.

[0159] Therapeutic Compositions

[0160] The differentially expressed polynucleotides and polypeptides ofthe invention may be used as therapeutic agents. Generally, therapeuticagents and methods of the invention are premised on modulating theactivity of an differentially expressed gene product (e.g.,polypeptide), where such modulation is accomplished by either enhancingor inhibiting gene expression or polypeptide activity. The active agentmay be a variety of different compounds, including a naturally occurringor synthetic small molecule compound, an antibody, fragment orderivative thereof, an antisense composition, and the like asexemplified below. Active agents described herein as therapeutic agentsmay also find use in non-therapeutic applications, e.g., in animalmodels of disease, as reagents, etc.

[0161] Where the therapeutic agent is used to decrease expression of thepolynucleotides of the invention, the agent can decrease expression ofthe polynucleotide by at least about 50%, usually at least about 60% to70%, and may facilitate a decrease in expression of at least about 80%or 90% to 95%, up to about 99% to 100%. The effectiveness of themechanism chosen to alter expression of the polynucleotide can beassessed using methods well known in the art, such as hybridization ofnucleotide probes to mRNA of the polynucleotide, quantitative RT-PCR, ordetection of a protein using specific antibodies of the invention.

[0162] Alternatively, agents for expression modulation can be designedto increase expression of a differentially expressed polynucleotide.Increasing expression of such polynucleotides may also be useful to, forexample, decrease the growth rate of pancreatic cancer cells and cancercells of other tissue origin, where the particular polynucleotide isdown-regulated in cancer cells. Within an expression construct, thepolynucleotide segment is oriented in the sense direction and is locateddownstream from the promoter. Transcription of the polynucleotidesegment initiates at the promoter. The expression construct can beintroduced into cells along with a pharmaceutically acceptable carrierto decrease the growth rate of cancer cells or ameliorate other abnormalcharacteristics. Expression of the polynucleotide sequence can bemonitored by detecting production of mRNA which hybridizes to thedelivered polynucleotide or by detecting protein encoded by thedelivered polynucleotide.

[0163] Exemplary agents for modulation of expression of differentiallyexpressed polynucleotides are provided below.

[0164] Small molecule compounds. Naturally occurring or synthetic smallmolecule compounds of interest include numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Active agents for modulation of activity alsoinclude antibodies that specifically bind a differentially expressedpolypeptide of the invention.

[0165] Antisense molecules. Antisense molecules can be used todown-regulate expression of genes in cells. The anti-sense reagent maybe antisense oligonucleotides (ODN), particularly synthetic ODN havingchemical modifications from native nucleic acids, or nucleic acidconstructs that express such anti-sense molecules as RNA. The antisensesequence is complementary to the mRNA of the targeted gene, and inhibitsexpression of the targeted gene products. Antisense molecules inhibitgene expression through various mechanisms, e.g. by reducing the amountof mRNA available for translation, through activation of RNAse H, orsteric hindrance. One or a combination of antisense molecules may beadministered, where a combination may comprise multiple differentsequences.

[0166] Antisense molecules may be produced by expression of all or apart of the target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844).

[0167] A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

[0168] Antisense oligonucleotides can be composed ofdeoxyribonucleotides, ribonucleotides, or a combination of both.Oligonucleotides can be synthesized manually or by an automatedsynthesizer, e.g., by covalently linking the 5′ end of one nucleotidewith the 3′ end of another nucleotide with phosphodiester ornon-phosphodiester internucleotide linkages such as alkylphosphonates,phosphorothioates, phosphorodithioates, alkylphosphonothioates,alkylphosphonates, phosphoramidates, phosphate esters, carbanates,acetamidate, carboxymethylesters, carbonates, and phosphate triesters.See Brown (1994) Meth. Mol. Biol. 20:1; Sonveaux (1994) Meth. Mol. Biol.26:1; Uhlmann et al. (1990) Chem. Rev. 90:543. Antisenseoligonucleotides may also be chemically synthesized by methods known inthe art (see Wagner et al. (1993), supra, and Milligan et al., supra.)

[0169] Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases. Among usefulchanges in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

[0170] Ribozymes. As an alternative to anti-sense inhibitors, catalyticnucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc. maybe used to inhibit gene expression. Ribozymes may be synthesized invitro and administered to the patient, or may be encoded on anexpression vector, from which the ribozyme is synthesized in thetargeted cell (for example, see International patent application WO9523225, and Beigelman et al. (1995), Nucl. Acids Res. 23:4434-42).Examples of oligonucleotides with catalytic activity are described in WO9506764. Conjugates of anti-sense ODN with a metal complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43-56. Ribozymesand their use to decrease expression of a selected gene is described in,for example, Cech (1987) Science 236:1532; Cech (1990) Ann. Rev.Biochem. 59:543; Cech (1992) Curr. Opin. Struct. Biol. 2:605; Couture etal. (1996) Trends Genet. 12:510; and U.S. Pat. No. 5,641,673). As perU.S. Pat. No. 5,641,673, ribozymes can be engineered so that theirexpression occurs in response to factors that also induce expression ofa polynucleotides of the invention. The ribozyme can also be engineeredto provide an additional level of regulation, so that destruction of RNAoccurs only when both the ribozyme and the corresponding gene areinduced in the cells.

[0171] Ribozymes of the invention can be introduced into cells as partof a DNA construct, as is known in the art. The DNA construct can alsoinclude transcriptional regulatory elements, such as a promoter element,an enhancer or UAS element, and a transcriptional terminator signal, forcontrolling the transcription of the ribozyme in the cells. Mechanicalmethods, such as microinjection, liposome-mediated transfection,electroporation, gene gun, or calcium phosphate precipitation, can beused to introduce the ribozyme-containing DNA construct into cells whosedivision it is desired to decrease, as described above. Alternatively,if it is desired that the DNA construct be stably retained by the cells,the DNA construct can be supplied on a plasmid and maintained as aseparate element or integrated into the genome of the cells, as is knownin the art.

[0172] Antibodies. Expression of the polynucleotides of the inventioncan also be decreased by delivering polyclonal, monoclonal, or singlechain antibodies that specifically bind to polypeptides expressed fromthe polynucleotide sequences as shown in SEQ ID NOS: 1-6. Antibodiesspecific to these proteins bind to the protein and prevent the proteinfrom functioning in the cell. Blocking protein expression or function isuseful for preventing, reducing the effects of, or curing pancreaticdisease.

[0173] Formulations. As mentioned above, an effective amount of theactive agent is administered to the host, where “effective amount” meansa dosage sufficient to produce a desired result, where the desiredresult in the desired modulation, e.g. enhancement, reduction, of thetarget protein activity.

[0174] The active agent can be incorporated into a variety offormulations for therapeutic administration. More particularly, theagents of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers, adjuvants, or diluents (e.g., liquids, such aswater, saline, glycerol, and ethanol), as well as substances such aswetting agents, emulsifying agents, or pH buffering agents, tonicityadjusting agents, stabilizers, wetting agents and the like. Liposomes,such as those described in U.S. Pat. No. 5,422,120, WO 95/13796, WO91/14445, or EP 524,968 B1, can also be used as a carrier for thetherapeutic composition. The compositions of the present invention maybe formulated into pharmaceutical preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.Pharmaceutically acceptable carriers are well known to those in the art.Such carriers include, but are not limited to, large, slowly metabolizedmacromolecule, such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andinactive virus particles. Pharmaceutically acceptable salts can also beused in the composition, for example, mineral salts such ashydrochlorides, hydrobromides, phosphates, or sulfates, as well as thesalts of organic acids such as acetates, proprionates, malonates, orbenzoates.

[0175] Administration and dosage. Administration of the agents can beachieved in various ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intratracheal, etc.,administration. In pharmaceutical dosage forms, the agents may beadministered in the form of their pharmaceutically acceptable salts, orthey may also be used alone or in appropriate association, as well as incombination, with other pharmaceutically active compounds. The methodsand excipients described herein are merely exemplary and are in no waylimiting.

[0176] The differentially expressed polynucleotides can be formulatedfor use in gene therapy to treat disorders associated with gene defects.Expression vectors, which generally have an expression cassette havingconvenient restriction sites located near a promoter sequence foroperable insertion of a polynucleotide of interest, may be used tointroduce the differentially expressed gene into a cell. The expressionvectors may be provided in a variety of forms (e.g. plasmid; retrovirus,e.g., lentivirus; adenovirus; and the like). Following introduction intothe cell, the vectors may be transiently or stably maintained either asan episome or as a genomic integrant, usually for a period of at leastabout one day, more usually for a period of at least about several daysto several weeks.

[0177] Typically, a therapeutic composition is prepared as aninjectable, either as a liquid solution or suspension; however, solidforms suitable for solution in, or suspension in, liquid vehicles priorto injection can also be prepared. A composition can also be formulatedinto an enteric coated tablet or gel capsule according to known methodsin the art, such as those described in U.S. Pat. No. 4,853,230, EP225,189, AU 9,224,296, and AU 9,230,801. In general, the agents can beformulated into preparations for injection by dissolving, suspending oremulsifying them in an aqueous or nonaqueous solvent, such as vegetableor other similar oils, synthetic aliphatic acid glycerides, esters ofhigher aliphatic acids or propylene glycol; and if desired, withconventional additives such as solubilizers, isotonic agents, suspendingagents, emulsifying agents, stabilizers and preservatives.

[0178] For oral preparations, the agents can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0179] The agents can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like. Furthermore,the agents can be made into suppositories by mixing with a variety ofbases such as emulsifying bases or water-soluble bases. The compounds ofthe present invention can be administered rectally via a suppository.The suppository can include vehicles such as cocoa butter, carbowaxesand polyethylene glycols, which melt at body temperature, yet aresolidified at room temperature.

[0180] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore inhibitors. Similarly, unit dosage forms for injection orintravenous administration may comprise the inhibitor(s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

[0181] The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

[0182] Kits with unit doses of a therapeutic agent of the invention,usually in oral or injectable doses, are provided. In such kits, inaddition to the containers containing the unit doses will be aninformational package insert describing the use and attendant benefitsof the drugs in treating pathological condition of interest.

[0183] Where the agent is a polypeptide, polynucleotide, analog ormimetic thereof, e.g. antisense composition, it may be introduced intotissues or host cells by any number of routes, including viralinfection, microinjection, or fusion of vesicles. Jet injection may alsobe used for intramuscular administration, as described by Furth et al.(1992), Anal. Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the DNA, then bombarded into skin cells.

[0184] Administration of the therapeutic agents of the invention caninclude systemic methods of delivery, as well as local administration.Various methods can be used to administer a therapeutic compositiondirectly to a specific site in the body. For treatment of tumors, forexample, a small tumor or metastatic lesion can be located and atherapeutic composition injected several times in several differentlocations within the body of the tumor. Alternatively, arteries whichserve a tumor can be identified, and a therapeutic composition injectedinto such an artery, in order to del! ver the composition directly intothe tumor.

[0185] A tumor which has a necrotic center can be aspirated and thecomposition injected directly into the now empty center of the tumor. Atherapeutic composition can be directly administered to the surface of atumor, for example, by topical application of the composition. X-rayimaging can be used to assist in certain of the above delivery methods.Combination therapeutic agents, including a polypeptide, polynucleotide,antibody, and/or other therapeutic agents, can be administeredsimultaneously or sequentially.

[0186] Receptor-mediated targeted delivery can be used to delivertherapeutic compositions containing polynucleotides, proteins,antibodies, ribozymes, or antisense oligonucleotides of the invention tospecific tissues. Receptor-mediated delivery techniques are describedin, for example, Findeis et al. (1993) Trends Biotechnol. 11:202; Chiouet al. (1994), Gene Therapeutics: Methods And Applications of DirectGene Transfer (J. A. Wolff, ed.); Wu et al. (1988), J. Biol. Chem.263:62; Wu et al., (1994) J. Biol. Chem. 269:542; Zenke et al. (1990)Proc. Natl. Sci. U.S.A. 87:3655; Wu et al. (1991) J. Biol. Chem.266:338.

[0187] Alternatively, therapeutic compositions can be introduced intohuman cells ex vivo, and the cells then replaced into the human. Cellscan be removed from a variety of locations including, for example, froma selected tumor or from an affected organ. In addition, a therapeuticcomposition can be inserted into non-affected cells, for example, dermalfibroblasts or peripheral blood leukocytes. If desired, particularfractions of cells such as a T cell subset or stem cells can also bespecifically removed from the blood (see, for example, PCT WO 91/16116).The removed cells can then be contacted with a therapeutic compositionutilizing any of the above-described techniques, followed by the returnof the cells to the human, preferably to or within the vicinity of atumor or other site to be treated. The methods described above canadditionally comprise the steps of depleting fibroblasts or othernon-contaminating tumor cells subsequent to removing tumor cells from ahuman, and/or the step of inactivating the cells, for example, byirradiation.

[0188] Both the dosage and means of administration can be determinedbased on a variety of factors such as the specific qualities of thetherapeutic composition, the condition, age, and weight of the patient,the progression of the disease, and other relevant factors. If thecomposition contains protein, polypeptide, or antibody, effectivedosages of the composition are in the range of about 5 pg to about 50mg/kg of patient body weight, about 50 pg to about 5 mg/kg, about 100 pgto about 500 mg/kg of patient body weight, and about 200 to about 250pg/kg. Those of skill in the art will readily appreciate that doselevels can vary as a function of the specific compound, the severity ofthe symptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound and for a given disease orcondition are readily determinable by those of skill in the art by avariety of means.

[0189] Therapeutic compositions for use in gene therapy applicationscontaining a differentially expressed polynucleotide of the inventioncan be administered in a range of about 100 ng to about 200 mg of DNAfor local administration in a gene therapy protocol. Concentrationranges of about 500 ng to about 50 mg, about 1 pg to about 2 mg, about 5pg to about 500 pg, and about 20 pg to about 100 pg of DNA can also beused during a gene therapy protocol. A variety of factors, such asmethod of action and efficacy of transformation and expression, willaffect the dosage required for ultimate efficacy. Where greaterexpression is desired over a larger area of tissue, larger amounts ofpolynucleotides or the same amounts re-administered in a successiveprotocol of administrations, or several administrations to differentadjacent or close tissue portions of, for example, a tumor site, can beused to effect a positive therapeutic outcome. In all cases, routineexperimentation in clinical trials will determine specific ranges foroptimal therapeutic effect.

[0190] Diseases amenable to therapy. The subject methods find use in thetreatment of a variety of different conditions involving differentiallyexpressed genes, including, for example, insufficient or hypo-proteinactivity and hyper-protein activity. In general, the subject methods andcompositions are useful where the modulation of expression or a functionof a differentially expressed gene product is desired. By “treatment” ismeant that at least an amelioration of the symptoms associated with thecondition afflicting the host, where amelioration is used in a broadsense to refer to at least a reduction in the magnitude of a parameter,e.g. symptom, associated with the condition being treated. As such,treatment also includes situations where the pathological condition, orat least symptoms associated therewith, are completely inhibited, e.g.prevented from happening, or stopped, e.g. terminated, such that thehost no longer suffers from the condition, or at least the symptoms thatcharacterize the condition.

[0191] A variety of hosts are treatable according to the subjectmethods. Generally such hosts are “mammals” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalian, including the orders carnivore (e.g., dogs and cats),rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g.,humans, chimpanzees, and monkeys), where “treatment” in the context ofnon-human hosts can involve the use of the subject compositions andmethods in animal models of disease. Of particular interest is thetreatment of human host.

[0192] Use of the therapeutic compositions and methods of the inventionin treating pancreatic cancer and pancreatic dysplasia is of particularinterest. The therapeutic compositions may also find use in treatment ofother types of cancers such as: bone cancer; brain tumors; breastcancer; endocrine system cancers, such as cancers of the thyroid,pituitary, and adrenal glands and the pancreatic islets;gastrointestinal cancers, such as cancer of the anus, colon, esophagus,gallbladder, stomach, liver, and rectum; genitourinary cancers such ascancer of the penis, prostate and testes; gynecological cancers, such ascancer of the ovaries, cervix, endometrium, uterus, fallopian tubes,vagina, and vulva; head and neck cancers, such as hypopharyngeal,laryngeal, oropharyngeal cancers, lip, mouth and oral cancers, cancer ofthe salivary gland, cancer of the digestive tract and sinus cancer;leukemia; lymphomas including Hodgkin's and non-Hodgkin's lymphoma;metastatic cancer; myelomas; sarcomas; skin cancer; urinary tractcancers including bladder, kidney and urethral cancers; and pediatriccancers, such as pediatric brain tumors, leukemia, lymphomas, sarcomas,liver cancer and neuroblastorna and retinoblastoma.

[0193] ATCC Depostits

[0194] The following plasmids were deposited as a bacterial culture withplasmid cDNA on Sep. 25, 1998 with the American Type Culture Collection,1301 Parklawn Drive, Rockville, Md., USA (ATCC) as ATCC accession no.98896:

[0195] 1) Clone HX2134-4 (containing an insert corresponding to SEQ IDNO: 1),

[0196] 2) Clone HX2144-1 (containing an insert corresponding to SEQ IDNO: 2);

[0197] 3) Clone HX2145-3 (containing an insert corresponding to SEQ IDNO: 3);

[0198] 4) Clone HX2162-3 (containing an insert corresponding to SEQ IDNO: 4);

[0199] 5) Clone HX2166-6 (containing an insert corresponding to SEQ IDNO: 5); and

[0200] 6) Clone HX2192-1 (containing an insert corresponding to SEQ IDNO: 6).

[0201] The deposit was made under the conditions specified by theBudapest Treaty on the international recognition of the deposit ofmicroorganisms (Budapest Treaty). Constructs and polynucleotidessequences equivalent to and/or substantially equivalent to the depositedmaterial are also considered to be within the scope of this invention.Availability of the deposited material is not to be construed as alicense to practice the invention in contravention of the rights grantedunder the authority of any government in accordance with its patentlaws.

[0202] Each of the above clones was transfected into separate bacterialcells, and were deposited as a pool of equal mixtures of all six clonesin this composite deposit. Each clone can be removed from the vector inwhich it was deposited by EcoRI to produce the appropriately sized 0.5kb-1.0 kb fragment for the clone. Particular clones can be obtained fromthe composite deposit using methods well known in the art. For example,a bacterial cell containing a particular clone can be identified byisolating single colonies on an appropriate bacterial media containingampicillin, and identifying colonies containing the specific clonethrough standard colony hybridization techniques, using anoligonucleotide probe or probes designed to specifically hybridize to asequence of one of SEQ ID NOS: 1-6. The probe should be designed to havea T_(m) of approximately 80° C. (assuming 2° C. for each A or T and 4°C. for each G or C). Positive colonies can then be picked, grown inculture, and the recombinant clone isolated.

EXAMPLES

[0203] The following examples are offered primarily for purposes ofillustration. It will be readily apparent to those skilled in the artthat the formulations, dosages, methods of administration, and otherparameters of this invention may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention.

Example 1

[0204] A family was identified that had several members who had beendiagnosed with pancreatic cancer. The family members also have a form ofdiabetes. The pathological features of disease in the family includedprogression from normal to metaplasia to dysplasia to cancer. Tissueswere obtained from a member of the family diagnosed with pancreaticcancer and from a member of the family diagnosed with dysplasia ofpancreatic cells, and primary cultures of ductal cells preparedaccording to methods well known in the art. Tissue was also obtainedfrom an unrelated person who was diagnosed with pancreatitis, and froman unrelated person who had a normal pancreas, and primary cultures ofductal cells prepared according to methods well known in the art.

[0205] The Genomyx HIEROGLYPH™ mRNA profile kit for differential displayanalysis was used according to the manufacturer's instructions toidentify genes that are differentially expressed in the various samplesrelative to one another. Briefly, mRNA was isolated from the primaryductal cell cultures, and subjected to reverse transcriptase polymerasechain reaction (PCR). The resulting cDNA was subjected to a differentialdisplay in which the cDNA from each of the samples were compared on agel.

[0206] The cDNA fragment pattern in each sample was manually compared tothe cDNA fragment pattern in every other sample on the gel. Those bandsrepresenting differentially expressed gene products (e.g., bandsassociated with relatively more or less cDNA in one sample relative toanother) were cut from the gel, amplified, cloned, and sequenced. Thefollowing polynucleotide sequences (SEQ ID NOS: 1-6) of cDNA fragmentsisolated from six such differentially displayed cDNA fragments wereidentified as being differentially regulated in pancreatic disease.TABLE 1 Results of Differential Display SEQ ID Clone Sequence Length NO.Name (bp) Results 1 HX2134-4 676 Expression decreased in dysplasia only2 HX2144-1 544 Expression increased in cancer only 3 HX2145-3 432Expression decreased in dysplasia only 4 HX2162-3 493 Expressionincreased in dysplasia only 5 HX2166-6 418 Expression increased indysplasia only 6 HX2192-1 1063 Expression decreased in dysplasia andcancer

[0207] The identification of these differentially expressedpolynucleotides, as well as the correlation of the relative levels ofexpression of the represented differentially expressed genes with thedisease states of pancreatic cancer and dysplasia, indicates that thegene products of the differentially expressed polynucleotides and genescan serve as markers of these disease states, where the markers can beused either singly or in combination with one another. Examination ofexpression of one or more of these differentially expressedpolynucleotides can thus be used in classifying the cell from which thepolynucleotides are derived as, for example, cancerous, dysplastic, ornormal, and can further be used in diagnosis of the subject from whomthe cell sample was derived. Use of all or a subset of thedifferentially expressed polynucleotides as markers will increase thesensitivity and the accuracy of the diagnosis.

Example 2

[0208] Sequencing and Analysis of Differentially ExpressedPolynucleotides

[0209] The sequences of the differentially expressed polynucleotidesidentified in Example 1 (SEQ ID NOS: 1-6) were used as query sequencesin the GenBank and dbEST public databases to identify possiblehomologous sequences. The search was performed using the BLAST program,with default settings. All six sequences were novel, i.e., no sequencepresent in the databases searched contained a sequence having thecontiguous nucleotide sequence set forth in any of SEQ ID NOS: 1-6.Moreover, each of the polynucleotides contained stretches of contiguousnucleotides for which no homologous sequence was identified. A summaryof these wholly unique sequences, referred to herein as identifyingsequences, is provided in Table 2 below. TABLE 2 Identifying sequencesof the differentially expressed genes of the invention. SEQ IDIdentifying Sequences NO: (numbering refers to nucleotide position inSequence Listing) 1 1-304; 533-571 2 1-62; 102-139; 183-544 3 1-41;62-182; 216-281; 319-432 4 1-13; 32-137; 156-236; 255-429; 453-493 51-101; 408-418 6 327-444; 640-997; 1018-1063

[0210] The identifying sequences above represent exemplary minimal,contiguous nucleotides sequences of the differentially expressedpolynucleotides than can be used in identification or detection of thecorresponding differentially expressed genes described herein.

Example 3

[0211] Fabricating a DNA Array Using Polynucleotides DifferentiallyExpressed in Pancreatic Cells

[0212] A DNA array is made by spotting DNA fragments onto glassmicroscope slides that are pretreated with poly-L-lysine. Spotting ontothe array is accomplished by a robotic arrayer. The DNA is cross-linkedto the glass by ultraviolet irradiation, and the free poly-L-lysinegroups are blocked by treatment with 0.05% succinic anhydride, 50%1-methyl-2-pyrrolidinone and 50% borate buffer.

[0213] The spots on the array are oligonucleotides synthesized on an ABIautomated synthesizer. Each spot is one of the polynucleotides of SEQ IDNOS: 1-6, each of which correspond to a gene that is differentiallyexpressed in pancreatic cells according to varying disease states (e.g.,overexpressed or underexpressed in cancerous, dysplastic, pancreatitis,and/or diabetic pancreatic cells). The polynucleotides may be present onthe array in any of a variety of combinations or subsets. Some internalstandards and negative control spots including non-differentiallyexpressed sequences and/or bacterial controls are included.

[0214] mRNA from patient samples is isolated, the mRNA used to producecDNA, amplified and subsequently labeled with fluorescent nucleotides asfollows: isolated mRNA is added to a standard PCR reaction containingprimers (100 pmoles each), 25 uM nucleotides, and 5 Units of Taqpolymerase (Perkin Elmer). In addition, fluorescent nucleotides(Cy3-dUTP (green fluorescence) or Cy5-dUTP (red fluorescence), sold byAmersham) are added to a final concentration of 60 uM. The reaction iscarried out in a Perkin Elmer thermocycler (PE9600) for 30 cycles usingthe following cycle profile: 92° C. for 30 seconds, 58° C. for 30seconds, and 72° C. for 2 minutes. Unincorporated fluorescentnucleotides are removed by size exclusion chromatography (Microcon-30concentration devices, sold by Amicon).

[0215] Buffer replacement, removal of small nucleotides and primers andsample concentration 25 is accomplished by ultrafiltration over anAmicon microconcentrator-30 (mwco=30,000 Da) with three changes of 0.45ml TE. The sample is reduced to 5 μl and supplemented with 1.4 μl 20×SSCand 5 μg yeast tRNA. Particles are removed from this mixture byfiltration through a pre-wetted 0.45μ microspin filter (Ultrafree-MC,Millipore, Bedford, Mass.). SDS is added to a 0.28% final concentration.The fluorescently-labeled cDNA mixture is then heated to 98° C. for 2min., quickly cooled and applied to the DNA array on a microscope slide.Hybridization proceeds under a coverslip, and the slide assembly is keptin a humidified chamber at 65° C. for 15 hours.

[0216] The slide is washed briefly in 1×SSC and 0.03% SDS, followed by awash in 0.06% SSC. The slide is kept in a humidified chamber untilfluorescence scanning was done. Fluorescence scanning and dataacquisition are then accomplished using any of a variety of suitablemethods well known in the art. For example, fluorescence scanning is setfor 20 microns/pixel and two readings are taken per pixel. Data forchannel 1 is set to collect fluorescence from Cy3 with excitation at 520nm and emission at 550-600 nm. Channel 2 collects signals excited at 647nm and emitted at 660-705 nm, appropriate for Cy5. No neutral densityfilters are applied to the signal from either channel, and thephotomultiplier tube gain is set to 5. Fine adjustments are then made tothe photomultiplier gain so that signals collected from the two spotsare equivalent.

[0217] The data acquired from the scan of the array is then converted toany suitable form for analysis. For example, the data may be analyzedusing a computer system, and the data may be displayed in a pictoralformat on a computer screen, where the display shows the array as acollection of spots, each spot corresponding to a location of adifferent polynucleotide on the array. The spots vary in brightnessaccording to the amount of fluorescent probe associated with the spot,which in turn is correlated with an amount of hybridized cDNA in thesample. The relative brightness of the spots on the array can becompared with one another to determine their relative intensities,either qualitatively or quantitatively.

[0218] The display of spots on the array, along with their relativebrightness, provides a test sample pattern. The test sample pattern canbe then compared with reference array patterns associated with positiveand negative control samples on the same array, e.g., an array havingpolynucleotides in substantially the same locations as the array usedwith the test sample. The reference array patterns used in thecomparison can be array patterns generated using samples from normalpancreas cells, cancerous pancreas cells, pancreatitis-associatedpancreas cells, diabetic pancreas cells, and the like. A substantial orsignificant match between the test array pattern and a reference arraypattern is indicative of a disease state of the patient from whom thetest sample was obtained.

[0219] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0220] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1 6 1 676 DNA Homo sapiens 1 agacaagagt ctcactctgt tgcccaggct ggagcacagcggtgcaatct tggctcacta 60 caacctccac ctcctgggtt taggcgattc tcctgcctcagcctcctaaa tagctgggat 120 tacaggcaca tgccaccaca cctggctaat ttttgtatttttagtagcga cggggtttcg 180 ccatgttggc caggctggtc tcgaactcct gacctcaggcgatccacttc ccgacctcag 240 gtgatccgcc tgcctcggcc tctaaaagtg ctgcaattacaggcataagc cactgtgcct 300 ggccctatcc cttttaattt tctaagtgac cagtaataaacaatgatttg tttattacta 360 ggtagcagga gaaaaaattt ttagtcactt ttccagctaagaatttcatt taaagatacc 420 tatgacatat cttgtggtac taagaatatt agagaactggaaatccagtt tttttgtggt 480 tttttaagaa agagaatctg actccattgc ccagcttggagagcagtggt gcaatagctg 540 gggctacagg cgtgagccac cacaccaggc ctggaaacccagttttaatt tgtgaactac 600 aaatggttgg caactgattc cttaattgtt attgcaggagtaggcccaac atgagtccat 660 atgtagccct tctctg 676 2 544 DNA Homo sapiens 2agggttgagg ctgggaacag ggagaacttc aaatgggcac aagaactcgt ttcggcatga 60tagaaatgtt ccaaaatgat actatggtaa tggtttcaca attcaaattt gctaaaactt 120actgagttgt gtacttaact gaattttata gtatgagaat tatatctcaa taaagctgta 180aacaaataaa ataactctat agaccttact gaaatagatg tcagttgcaa ggcatcatct 240cccatttcct gtgcaattct atctccttta catttgaaag gcttgagtca caccagccag 300ctggtggtct ttgatccccg ctctgggtcc cttaccctaa agaaaggact tgagcacttt 360ctaagggtaa actctctgga gactttttca gaaaggtctc aacaagggtc cacttattct 420cggggaagct cataaaagaa acattattgg tctcttgaaa gttcaaaagg gccattttat 480gagatgagaa tgaatgactt tccattcttt cttgcttttc attgtagaag tgacttacat 540taag 544 3 432 DNA Homo sapiens 3 gggtaggata tggctatgtg actttctaagctatagctat ggaaaacatt gaaacaaaat 60 tcacgcatgc cgtgtactgt gcccagaaattgtagacttg tctgggtggt gttaaggatt 120 tgacctattg agagtactca cacctgcttcttacattcaa gttgttaatc cttcgttcag 180 aaaaggagac atattaggaa atatgtgaatggactgctgt gacttaaatc ttaagtgttt 240 aggctcttaa actggttcaa actaaaataacattgatgaa atagtgtttt agacttctgt 300 tacatttatt atatttcaga aacctgtgtgagttgacttt actctcattt aagatctagg 360 actttgttgg aacgcttgcc tgagttctaagttgtaacaa agggtcctga aaatatttag 420 ttacccaaaa tt 432 4 493 DNA Homosapiens 4 atgaactcgg tttaagacag ggcttcttca ccattgcgag aacgttcaccgggacgagtg 60 gcaagagtct tggcttggat agcatgaaga gccccagtac aaggaagaatactggaaatg 120 ctcaattcgt ggagcgcgtt taaacgacga tttatttggt tttcaatgatcgaggactta 180 tgacaggatg attacatttg accttgggac atgaacgctt ggactgctgacttgtgtgta 240 aagctgtttt gcttgtttgt gtcttgcttg acagtggttc tcgatcatgatgatacctga 300 tgctttggac atgtccactt actcctctat tattcgttgg atcattgtttattctgatag 360 atagtgactt atgttcggat gtcgatcaca ggattgtgat tgttagtccactgtatctct 420 gatcgaatag gtctatatat tattatttag atagaaaaag tagcaatccacttaggagat 480 ttattgatct gct 493 5 418 DNA Homo sapiens 5 ctattcctatctaatgctag tcttcccact tgggcacttg attcccctct tgcatactca 60 agggcttggcttctatagct acccctttca tttgcatcgt tagttttttc acttgcaata 120 atttactatcagccctcttg gccaggtgca gtggctcacc cgtcatccca gcactttggg 180 aggccaaagtgggtggatca cttgaggtcg gaagttcgag accagcctgg ccaacatggc 240 aaaaccctgtctctactaaa agtacaaaaa ttagccagga atggtagcac atgcctgtaa 300 tcccagctactcgggaggct gaggcagaat cgcttgagcc caggaggcgg aggttgcagt 360 gagccaagatggcaccactg cactccagcc tgggcaacag agcaagaaga ctccatct 418 6 1063 DNA Homosapeins 6 ccaattttcc ccttttggaa tgggaatgtt tacccaatgt ctatacccccattgtatctt 60 gggagtaaat aagttattac agggtcatag gtggaaggaa ctcatcttcttcagataaga 120 ttttggactt tggagttaat gttgaaataa gttaagactt taggggactgttaagaagag 180 attattgtat tttgtgatgt gagaaagaca ggagatttaa gggggccagaggcagaagga 240 aatagtttgt atatttgtcc ctgcccaaat ttcacattga aatgtaatccccaatgttgg 300 tggtagggcc tagtgggaag tgtttgtctg atggtggcag atccatgatgaatgacttgg 360 taccattaat ttggtgatga gttgtctttc tcacttcaca tatccagttgtttaaaagtg 420 tggggtccct ccccccaacc cctgccttgc tcctgctttc acaatgtgaagcgcctgctc 480 ccacttcact ttccgccatg agtaaaagct ccttgaggcc ttcccagaagctaagcagat 540 gccagagcac catgcttcct gtacaacctg cagaatcatg agccaattaaaactcttttc 600 tttataaatt acccagtctc agatatttct ttatagcaag aatggcctactacacaaaat 660 tggtattgta tgaactgtac tccggttgga gtaaatttgt ttttgacacaagcgtaggtt 720 agtagctctg aagctatatt aaatgtatac tagagttgaa caaataataaattgtagata 780 atgagagcca gattacaaga aagaagtgac aggtaagcca ggggggaaggctagaatgaa 840 ctgtgtggca cgggattagt gttcgcaata tcacaagaat gcacatttatgcaatacaga 900 aatagagata tgtgagtata catggtgaag tatacataca tatatgaggggacctaggag 960 caatgatacc ttgggagcaa tgaacacacc taaagcccag atcttggtttctaaatacta 1020 ttctccaata aaagagtcta aggtctttaa agaaatggtt tat 1063

What is claimed is:
 1. A library of differentially expressed genes, thelibrary comprising the sequence information of at least one of thepolynucleotides of SEQ ID NOS: 1-6.
 2. The library of claim 1, whereinthe library is provided on a nucleic acid array.
 3. The library of claim1, wherein the library is provided in a computer-readable format.
 4. Thelibrary of claim 1, wherein the library is composed of relative amountsof the polynucleotides of SEQ ID NOS: 1-6, where the relative amounts isrepresentative of relative amounts of the polynucleotides found in acancerous pancreatic cell.
 5. The library of claim 1, wherein thelibrary is composed of relative amounts of polynucleotides of SEQ IDNOS: 1-6, where the relative amounts is representative of relativeamounts of the polynucleotides found in a dysplastic pancreatic cell. 6.An isolated polynucleotide comprising a nucleotide sequence having atleast 90% sequence identity to an identifying sequence of SEQ ID NOS:1-6 or degenerate variants thereof.
 7. The isolated polynucleotide ofclaim 6, wherein the identifying sequence is selected from the groupconsisting of: a) residues 1-304 of SEQ ID NO: 1; b) residues 533-571 ofSEQ ID NO: 1; c) residues 1-62 of SEQ ID NO: 2; d) residues 102-139 ofSEQ ID NO: 2; e) residues 183-544 of SEQ ID NO: 2; f) residues 1-41 ofSEQ ID NO: 3; g) residues 62-182 of SEQ ID NO: 3; h) residues 216-281 ofSEQ ID NO: 3; i) residues 319-342 of SEQ ID NO: 3; j) residues 1-13 ofSEQ ID NO: 4; k) residues 32-137 of SEQ ID NO: 4; 1) residues 156-236 ofSEQ ID NO: 4; m) residues 255-429 of SEQ ID NO: 4; n) residues 453-493of SEQ ID NO: 4; o) residues 1-101 of SEQ ID NO: 5; p) residues 408-418of SEQ ID NO: 5; q) residues 327-444 of SEQ ID NO: 6; r) residues640-997 of SEQ ID NO: 6; and s) residues 1018-1063 of SEQ ID NO:
 6. 8.An array comprising the polynucleotide of claim
 6. 9. A recombinant hostcell containing the polynucleotide of claim
 6. 10. An isolatedpolypeptide encoded by the polynucleotide of claim
 6. 11. An antibodythat specifically binds a polypeptide of claim
 10. 12. A polynucleotidecomprising the nucleotide sequence of an insert contained in a cloneselected from the group consisting of: a) clone HX2134-4, deposited asATCC accession number 98896; b) clone HX2144-1, deposited as ATCCaccession number 98896; c) clone HX2145-3, deposited as ATCC accessionnumber 98896; d) clone HX2162-3, deposited as ATCC accession number98896; e) clone HX2166-6, deposited as ATCC accession number 98896; andf) clone HX2192-1, deposited as ATCC accession number
 98896. 13. Amethod of identifying a cancerous pancreatic cell, the method comprisingthe step of: detecting at least one differentially expressed geneproduct, where the gene product is encoded by a gene having a sequenceof SEQ ID NOS: 1-6 in a test sample, where the test sample is derivedfrom a test cell suspected of being a cancerous pancreatic cell; andcomparing an amount of the detected differentially expressed geneproduct with an amount of the differentially expressed gene product in acontrol sample, where the control sample is derived from a cancerouspancreatic cell; wherein detection of an amount of the differentiallyexpressed gene product in the test sample that is similar to an amountof the gene product in the control sample indicates that the test cellsis a cancerous pancreatic cell.
 14. The method of claim 13, wherein saiddetecting step is by hybridization of the test sample to a referencearray, wherein the reference array comprises an identifying sequence ofat least one of SEQ ID NOS: 1-6.
 15. A therapeutic compositioncomprising an active agent for modulation of expression of a genedifferentially expressed in cancerous or dysplastic pancreatic cells.16. The therapeutic composition of claim 15, wherein the active agenteffects a decrease in biological activity of a gene product encoded by agene having a sequence of SEQ ID NO:
 2. 17. The therapeutic compositionof claim 15, wherein the active agent effects an increase in biologicalactivity of a gene product encoded by a gene having a sequence of SEQ IDNO:
 6. 18. The therapeutic composition of claim 15, wherein the activeagent effects an increase in biological activity of a gene productencoded by a gene having a sequence of SEQ ID NOS: 1 or
 3. 19. Thetherapeutic composition of claim 15, wherein the active agent effects adecrease in biological activity of a gene product encoded by a genehaving a sequence of SEQ ID NOS: 4 or 5.